Anti-IL13 antibodies and uses thereof

ABSTRACT

The present invention relates to anti-IL13 antibodies that bind specifically and with high affinity to both glycosylated and non-glycosylated human IL13, does not bind mouse IL13, and neutralize human IL13 activity at an approximate molar ratio of 1:2 (MAb:IL13). The invention also relates to the use of these antibodies in the treatment of IL13-mediated diseases, such as allergic disease, including asthma, allergic asthma, non-allergic (intrinsic) asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergies, chronic obstructive pulmonary disease, ulcerative colitis, RSV infection, uveitis, scleroderma, and osteoporosis.

This application is a divisional of U.S. application Ser. No.10/583,927, filed Jan. 29, 2009, now U.S. Pat. No. 8,067,199 which is anational stage of International Application No. PCT/US2004/043501 filedDec. 23, 2004, which claims the benefit of U.S. Provisional ApplicationNo. 60/532,130 filed Dec. 23, 2003; each of which is incorporated byreference herein in its entirety.

BACKGROUND OF INVENTION

The interleukin (IL)-13 is a pleiotropic T helper cell subclass 2 (Th2)cytokine. Like IL4, IL13 belongs to the family of type I cytokinessharing the tertiary structure defined by a 4α-helical hydrophobicbundle core. IL13 has approximately 30% amino acid sequence homologywith IL4 and shares many of the properties of IL4 (Wynn, Ann. Rev.Immunol., 21: 425 (2003)). The functional similarity of IL4 and IL13 isattributed to the fact that IL13 can bind IL4 receptor alpha chain(IL4R-α) subsequent to its binding to IL13 receptor alpha chain-1(IL13Rα1) (Hershey, J. Allergy Clin. Immunol., 111: 677 (2003)). IL4Rαis activated by IL4 and IL13 resulting in Jak1-dependent STAT6phosphorylation. Both IL4 and IL13 promote B-cell proliferation andinduce class switching to IgG4 and IgE in combination with CD40/CD40Lcostimulation (Punnonen et al., Proc. Natl. Acad. Sci. USA, 90: 3730(1993), Oettgen et al., J. Allergy Clin. Immunol., 107: 429 (2001)).

However, unlike IL4, IL13 is not involved in the differentiation ofnaïve T cells into Th2 cells (Zurawski et al., Immunol. Today, 15: 19(1994)). IL13 up-regulates FcεRI and thus helps in IgE priming of mastcells (de Vries, Allergy Clin. Immunol. 102: 165 (1998). Inmonocytes/macrophages, IL13 up-regulates expression of CD23 and MHCclass I and class II antigens, down-regulate the expression of Fcγ andCD14, and inhibit antibody-dependent cytotoxicity (de Waal Malefyt etal., J. Immunol., 151: 6370 (1993), Chomarat et al., Int. Rev. Immunol.,17: 1 (1998)). IL13, but not IL4, promotes eosinophil survival,activation, and recruitment (Horie et al., Intern. Med., 36: 179 (1997),Luttmann et al., J. Immunol. 157: 1678 (1996), Pope et al., J. AllergyClin. Immunol., 108: 594 (2001). IL13 also manifests important functionson nonhematopoietic cells, such as smooth muscle cells, epithelialcells, endothelial cells and fibroblast cells. IL13 enhancesproliferation and cholinergic-induced contractions of smooth muscles(Wills-Karp, J. Allergy Clin. Immunol., 107: 9 (2001). In epithelialcells IL13 is a potent inducer of chemokine production (Li et al., J.Immunol., 162: 2477 (1999), alters mucociliary differentiation (Laoukiliet al., J. Clin. Invest., 108: 1817 (2001), decreases ciliary beatfrequency of ciliated epithelial cells (Laoukili et al., J. Clin.Invest., 108: 1817 (2001), and results in goblet cell metaplasia (Zhu etal., J. Clin. Invest., 103: 779 (1999), Grunig et al., Science, 282:2261 (1998)). In endothelial cells IL13 is a potent inducer of vascularcell adhesion molecule 1 (VCAM-1) which is important for recruitment ofeosinophils (Bochner et al., J. Immunol., 154: 799 (1995)). In humandermal fibroblasts IL13 induces type 1 collagen synthesis in humandermal fibroblasts (Roux et al., J. Invest. Dermatol., 103: 444 (1994)).

Although IL13 and IL4 share certain functional similarities, studies inanimal models of disease and gene-knockout mice demonstrated that IL13possesses unique effector functions distinct from IL4 and providescompelling evidence that IL13, independent of other Th2 cytokines, isnecessary and sufficient to induce all features of allergic asthma(Wills-Karp et al. Science, 282: 2258 (1998), Walter et al. J. Immunol.167: 4668 (2001)). IL13 may play a more significant role than other Th2cytokines in effector functions associated with the symptoms of asthma(Corry, Curr. Opin. Immunol., 11: 610 (1999)). This contention issupported in human disease by a strong association between IL13 levelsand genetic polymorphisms in the IL13 gene and disease correlates(Wills-Karp. et al. Respir. Res. 1: 19 (2000); Vercelli et al., Curr.Opin. Allergy Clin. Immunol., 2: 389 (2002); He et al., Genes Immunol.,4: 385 (2003), Arima et al., J. Allergy Clin. Immunol., 109: 980 (2003),Liu et al., J. Clin. Allergy Immunol., 112: 382 (2003)). Emerging datasuggest that IL13 induces features of the allergic response via itsactions on mucosal epithelium and smooth muscle cells, rather thanthrough the traditional pathways involving eosinophils and IgE-mediatedevents (Wills-Karp et al., Sci., 282: 2258 (1998)).

Asthma is described as a chronic pulmonary disease that involves airwayinflammation, hyperresponsiveness and obstruction. Physiologically,airway hyperresponsiveness is documented by decreased bronchial airflowafter bronchoprovocation with methacholine or histamine. Other triggersthat provoke airway obstruction include cold air, exercise, viral upperrespiratory infection, cigarette smoke, and respiratory allergens.Bronchial provocation with allergen induces a prompt early phaseimmunoglobulin E (IgE)-mediated decrease in bronchial airflow followedin many patients by a late-phase IgE-mediated reaction with a decreasein bronchial airflow for 4-8 hours. The early response is caused byacute release of inflammatory substances, such as histamine, PGD₂,leukotriene, tryptase and platelet activating factor (PAF), whereas thelate response is caused by de novo synthesized pro-inflammatorycytokines (e.g. TNFα, IL4, IL13) and chemokines (e.g. MCP-1 and MIP-1α)(Busse et al. In: Allergy: Principles and Practice, Ed. Middleston, 1173(1998)). In chronic asthmatic patients, persistent pulmonary symptomsare mediated by the heightened response of Th2 cells. Th2 cytokines arebelieved to play a vital role in the disease (Larche et al., J. AllergyClin. Immunol., 111: 450 (2003)), in particular, IL13 and IL4 producedby Th2 cells with NK phenotype (NKT) in the airway as indicated in amodel of asthma in rodents (Akbari et al., Nature Med., 9: 582 (2003)).The gross pathology of asthmatic airways displays lung hyperinflation,smooth muscle hypertrophy, lamina reticularis thickening, mucosal edema,epithelial cell sloughing, cilia cell disruption, and mucus glandhypersecretion. Microscopically, asthma is characterized by the presenceof increased numbers of eosinophils, neutrophils, lymphocytes, andplasma cells in the bronchial tissues, bronchial secretions, and mucus.Initially, there is recruitment of leukocytes from the bloodstream tothe airway by activated CD4+ T-lymphocytes. The activated T-lymphocytesalso direct the release of inflammatory mediators from eosinophils, mastcells, and lymphocytes. In addition, the Th2 cells produce IL4, IL5, IL9and IL13. IL4, in conjunction with IL13, signals the switch from IgM toIgE antibodies.

Cross-linking of membrane-bound IgE molecules by allergen causes mastcells to degranulate, releasing histamine, leukotrienes, and othermediators that perpetuate the airway inflammation. IL5 activates therecruitment and activation of eosinophils. The activated mast cells andeosinophils also generate their cytokines that help to perpetuate theinflammation. These repeated cycles of inflammation in the lungs withinjury to the pulmonary tissues followed by repair may produce long-termstructural changes (“remodeling”) of the airways.

Moderate asthma is currently treated with a daily inhaledanti-inflammatory-corticosteroid or mast cell inhibitor such as cromolynsodium or nedocromil plus an inhaled beta2-agonist as needed (3-4 timesper day) to relieve breakthrough symptoms or allergen- orexercise-induced asthma. Cromolyn sodium and nedocromil blockbronchospasm and inflammation, but are usually effective only for asthmathat is associated with allergens or exercise and typically, only forjuvenile asthmatics. Inhaled corticosteroids improve inflammation,airways hyperreactivity, and obstruction, and reduce the number of acuteexacerbations. However, it takes at least a month before effects areapparent and up to a year for marked improvement to occur. The mostfrequent side effects are hoarseness and oral fungal infection, i.e.,candidiasis. More serious side effects have been reported, e.g., partialadrenal suppression, growth inhibition, and reduced bone formation, butonly with the use of higher doses. Beclomethasone, triamcinolone, andflunisolide probably have a similar potency; whereas budesonide andfluticasone are more potent and reportedly have fewer systemic sideeffects.

Even patients with mild disease show airway inflammation, includinginfiltration of the mucosa and epithelium with activated T cells, mastcells, and eosinophils. T cells and mast cells release cytokines thatpromote eosinophil growth and maturation and the production of IgEantibodies, and these, in turn, increase microvascular permeability,disrupt the epithelium, and stimulate neural reflexes andmucus-secreting glands. The result is airways hyperreactivity,bronchoconstriction, and hypersecretion, manifested by wheezing,coughing, and dyspnea.

Traditionally, asthma has been treated with oral and inhaledbronchodilators. These agents help the symptoms of asthma, but donothing for the underlying inflammation. Recognition during the last 10years of the importance of inflammation in the etiology of asthma hasled to the increased use of corticosteroids, but many patients continueto suffer from uncontrolled asthma.

Because of the importance of treating inflammatory diseases in humans,particularly asthma, new bioactive compounds having fewer side effectsare continually being sought. The development of potent and specificinhibitors of IL13, which remain active when administered long term toasthmatic airways, offers a novel approach to the treatment of asthma,as well as in other IL13- and IgE-mediated diseases.

SUMMARY OF INVENTION

The present invention relates at least in part to antibodies that bindspecifically and with high affinity to both glycosylated andnon-glycosylated human IL13; does not bind mouse IL13, and neutralizehuman IL13 activity at an approximate molar ratio of 1:2 (MAb:IL13).Also included in the present invention are antibodies comprising theantigen binding regions derived from the light and/or heavy chainvariable regions of said antibodies. The antibodies of the invention maybe monoclonal, and a monoclonal antibody may be a human antibody, achimeric antibody, or a humanized antibody.

Examples of these antibodies are 228B/C-1, 228A-4, 227-26, and 227-43.The hybridomas that produce these antibodies were deposited on Nov. 20,2003, with the American Type Culture Collection, 10801 University Blvd.,Manassas, Va. 20110-2209, under Accession Numbers PTA-5657, PTA-5656,PTA-5654, and PTA-5655, respectively.

The present invention includes antibodies which have a VL sequence atleast 95% homologous to that set forth in SEQ ID NO: 3, and a VHsequence at least 95% homologous to that set forth in SEQ ID NO: 4;antibodies which have a VL sequence at least 95% homologous to that setforth in SEQ ID NO: 5, and a VH sequence at least 95% homologous to thatset forth in SEQ ID NO: 6; and antibodies which have a VL sequence atleast 95% homologous to that set forth in SEQ ID NO: 7, and a VHsequence at least 95% homologous to that set forth in SEQ ID NO: 8. Thepresent invention also includes a recombinant antibody molecule, or anIL13-binding fragment thereof, comprising at least one antibody heavychain, or an IL13-binding fragment thereof, comprising non-human CDRs atpositions 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) (Kabat numbering)from a mouse anti-IL13 antibody, wherein positions 27-30 have the aminoacid Gly 26, Phe 27, Ser 28, Leu 29, Asn 30, (SEQ ID NO: 18); and atleast one antibody light chain, or an IL13-binding fragment thereof,comprising non-human CDRs at positions 24-34 (CDR1), 50-56 (CDR2) and89-97 (CDR3) from a mouse anti-IL13 antibody, and framework regions froma human monoclonal antibody.

The present invention includes human antigen-binding antibody fragmentsof the antibodies of the present invention including, but are notlimited to, Fab, Fab′ and F(ab′)₂, Fd, single-chain Fvs (scFv),single-chain antibodies, disulfide-linked Fvs (sdFv). The invention alsoincludes single-domain antibodies comprising either a VL or VH domain.On example of an scFv is depicted in FIG. 21, having the sequence of SEQID NO 152.

The present invention includes humanized sequences of monoclonalantibody 228B/C-1. These humanized recombinant antibody moleculescomprise a variable light chain region comprising an amino acid sequencehaving the formula: FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, wherein FRL1consists of any one of SEQ ID Nos: 20-25; CDRL1 consists of any one ofSEQ ID NOs: 99-103; FRL2 consists of SEQ ID NO: 29; CDRL2 consists ofany one of SEQ ID NOs: 104-114; FRL3 consists of any one of SEQ ID NOs:30-56; CDRL3 consists of any of SEQ ID NOs: 115-116; and FRL4 consistsof SEQ ID NO: 57-59; and comprising a variable heavy chain regioncomprising an amino acid sequence having the formula:FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, wherein FRH1 consists of any oneof SEQ ID NOs: 60-66; CDRH1 consists of any one of SEQ ID NOs: 117-122;FRH2 consists of any one of SEQ ID NOs: 67-75; CDRH2 consists of any oneof SEQ ID NOs: 123-134; FRH3 consists of any one of SEQ ID NOs: 76-90;CDRH3 consists of any of SEQ ID NOs: 135-141; and FRH4 consists of SEQID NO: 91-92. The variable heavy chain region may further comprise atleast the CH1 domain of a constant region or the CH1, CH2 and CH3domains of a constant region. The heavy chain constant region maycomprise an IgG antibody. wherein the IgG antibody is an IgG1 antibody,an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

The present invention also includes recombinant antibody moleculeswherein the variable light chain is chosen from any one of SEQ ID Nos:3, 5, 7, 93, 95, 97, 142, 144, and 150, and a variable heavy chainchosen from any one of SEQ ID Nos: 4, 6, 8, 94, 96, 98, 143, 145, 146,147, 148, and 149. One particular antibody comprises the variable lightchain having the sequence set forth in SEQ ID NO:142, and a variableheavy chain having the sequence set forth in SEQ ID NO:143.

The present invention includes the hybridoma cell lines that produce themonoclonal antibodies 228B/C-1, 228A-4, 227-26, and 227-43. The presentinvention includes nucleic acids encoding the monoclonal antibodies228B/C-1, 228A-4, 227-26, and 227-43, cell lines comprising a nucleicacid encoding these antibodies or chains thereof, and vectors comprisingthe nucleic acid encoding these antibodies or chains thereof.

The present invention also includes antibodies that bind the sameepitope as 228B/C-1. Exemplary polypeptides comprise all or a portion ofSEQ ID NO. 1 or variants thereof, or SEQ ID NO. 2, wherein amino acid 13is changed from glutamic acid to lysine. The invention also relates tothe epitope recognized by the antibodies of the present invention.Epitope peptides include a peptide comprising essentially or consistingof ESLINVSG (SEQ ID NO: 18) or YCAALESLINVS (SEQ ID NO:19).

The present invention includes a composition comprising the antibodiesaccording to the claimed invention in combination with apharmaceutically acceptable carrier, diluent, excipient, or stabilizer.

The present invention includes a method of treating a subject sufferingfrom asthmatic symptoms comprising administering to a subject, e.g. asubject in need thereof, an amount of an antibody according to theclaimed invention effective to reduce the asthmatic symptoms, whereinthe antibody may down-regulate the activity of IL13 in the patient,reduce bronchial hyperresponsiveness in the patient, and/or reduceeosinophilia in the lungs of the subject. The present invention alsoincludes a method of inhibiting the infection of respiratory syncytialvirus (RSV) comprising administering to a subject, e.g. a subject inneed thereof, an inhibiting amount of the antibody of the claimedinvention.

The antibody of the present invention may be administered by one or moreof the routes including intravenous, intraperitoneal, inhalation,intramuscular, subcutaneous and oral routes. The present inventionincludes an inhalation device that delivers to a patient atherapeutically effective amount of an antibody according to the claimedinvention.

The present invention includes a method for detecting interleukin-13protein in a subject, e.g., a patient suffering from an allergicdisease, comprising, e.g., the steps of allowing the antibody of theclaimed invention to contact a sample; and detecting the interleukin-13through the occurrence of immunoreaction. Also described are and methodsfor diagnosing overexpression of IL13 in a subject, comprising the stepsof (a) obtaining a sample from the subject; (b) combining the samplewith an antibody according to the claimed invention under conditionswhich would allow immunoreaction with IL13; and (c) determining whetheror not IL13 is overexpressed relative to a normal level of expression ofIL13.

The present invention includes a method for producing the antibodies ofthe claimed invention, comprising the steps of: a) producing animmunogenic compound comprising a glycosylated IL13 moiety and animmunogenic moiety; b) preparing an injectable solution comprising saidimmunogenic compound in phosphate buffered saline (PBS) and an adjuvant;c) immunizing a mouse with said injectable solution by a combination ofintravenous and intraperitoneal injections, d) producing a hybridoma byfusing a spleen cell from said immunized mouse with a myeloma cell; e)selecting a hybridoma producing an antibody having the characteristicsof the antibody of the claimed invention; and f) isolating saidantibody.

The present invention includes a method for inhibiting IgE antibodyproduction in a patient, which comprises administrating to the patientan effective amount of an IgE antibody production inhibiting effectiveamount of an antibody according to the claimed invention. The inhibitionof IgE antibody production may prevent bronchial asthma, allergicrhinitis, allergic dermatitis, and anaphylaxis, and also treat bronchialasthma, allergic rhinitis, uticaria, and atopic dermatitis.

The present invention includes a method of treating an IL13-mediateddisorder in a patient, comprising administering to the patient aneffective amount of an antibody or antigen-binding fragment thereofaccording to the claimed invention, wherein said antibody orantigen-binding fragment thereof inhibits binding of IL13 to itsreceptor and inhibits one or more functions associated with binding ofthe interleukin to said receptor.

The present invention includes a method of treating an IgE-mediateddisorder in a patient, comprising administering to the patient aneffective amount of an antibody or antigen-binding fragment thereofaccording to the claimed invention, wherein said antibody orantigen-binding fragment thereof inhibits binding of IL13 to itsreceptor and inhibits one or more functions associated with binding ofthe interleukin to said receptor.

The present invention includes a method for reducing the severity ofasthma in a mammal comprising administering to the mammal atherapeutically effective amount of an anti-IL13 monoclonal antibodyhaving at least one of the following characteristics: the ability tobind human IL13 with a K_(D) between about 1×10¹⁰ to about 1×10¹² M; theability to inhibit one or more functions associated with binding of theinterleukin IL13 to the IL13 receptor; and the inability of the antibodydoes to bind to mouse IL13.

Diseases and/or conditions mediated by IL13 that are contemplated by theinvention include, but are not limited to, allergic asthma, non-allergic(intrinsic) asthma, allergic rhinitis, atopic dermatitis, allergicconjunctivitis, eczema, urticaria, food allergies, chronic obstructivepulmonary disease, ulcerative colitis, RSV infection, uveitis,scleroderma, and osteoporosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the binding of anti-IL13 monoclonal antibodies to humanIL13.

FIG. 2 depicts the binding of anti-IL13 monoclonal antibodies mutantIL13-Fc.

FIG. 3 illustrates that there is no inhibition of MAb 228B/C-1 bindingto human IL13 by MAb JES10-5A2 (Pharmingen).

FIG. 4 illustrates the effect of anti-IL13 monoclonal antibodies on theproliferation of Hodgkin Lymphoma L-1236 cells.

FIG. 5 illustrates the effect of anti-IL13 monoclonal antibodies onIL13-induced suppression of CD14 expression in human monocytes.

FIG. 6 illustrates the effect of anti-IL13 monoclonal antibodies onIL13-induced up-regulation of CD23 expression in human monocytes.

FIG. 7 illustrates the effect of anti-IL13 monoclonal antibodies onIL13-induced STAT6 phosphorylation in THP-1 cells.

FIG. 8 depicts the amino acid sequence of the VH and VL regions ofmonoclonal antibody 228B/C-1.

FIG. 9 depicts the amino acid sequence of the VH and VL regions ofmonoclonal antibody 228A-4.

FIG. 10 depicts the amino acid sequence of the VH and VL regions ofmonoclonal antibody 227-26.

FIGS. 11A-1, 11A-2, 11B-1, 11B-2, 11C-1, 11C-2, 11D-1, and 11D-2 depictthe sequences of the light chain variable regions (VK) for humanizationof monoclonal antibody 228B/C-1. Clones B to R represent clones testedwith a human template 2 for VK and a murine VH. HT2-NEW and HT2-DP27clones were constructed with human frameworks for both VK and VH. Theamino acid sequences of framework region (FR) 1 (FIGS. 11A-1 and 11C-1),FR2 (FIGS. 11A-2 and 11C-2), FR3 (FIGS. 11B-1 and 11D-1), and FR4 (FIGS.11B-2 and 11D-2) of the VK of the indicated clones are depicted.

FIGS. 12A-1, 12A-2, 12B-1, 12B-2, 12C-1, 12C-2, 12C-3, 12C-4, 12D-1,12D-2, 12D-3, and 12D-4 depict the corresponding heavy chain variableregion sequences of clones in FIG. 11. (i.e., 11A-1, 11A-2, 11B-1,11B-2, 11C-1, 11C-2, 11D-1, and 11D-2). The amino acid sequences of FR1(FIGS. 12A-1, 12C-1, and 12C-2), FR2 (FIGS. 12A-2, 12C-3, and 12C-4),FR3 (FIGS. 12B-1, 12D-1 and 12D-2), and FR4 (FIGS. 12B-2, 12D-3 and12D-4) of the VH of the indicated clones are depicted.

FIG. 13 A-D depict ELISA profiles for combinatorial humanizedcandidates.

FIG. 14 A depicts ELISA profiles for 89 Vk/276G. FIG. 14B depicts theELISA results for construct 115Vk/73Vh FL.

FIG. 15 depicts the sequences of combinatorial library candidates.

FIG. 16 depicts a competition profile for two candidates (CL5 and CL-13)assayed demonstrated as compared with the chimeric candidate (228 B/C#3) for binding to IL-13. The irrelevant Fab is 5I, which demonstratesno ability to compete.

FIG. 17 depicts the sequences of three affinity matured candidates.

FIG. 18 shows the alignment of IL13 protein sequences. The amino acidsequence for the following species of IL-13 protein are aligned: human(SEQ ID NO: 187), monkey (SEQ ID NO: 188), bovine (SEQ ID NO: 189), dog(SEQ ID NO: 190), rat (SEQ ID NO: 191), mouse (SEQ ID NO: 192), andgerbil (SEQ ID NO: 193). The majority sequence (SEQ ID NO: 186) based onthe alignment is depicted.

FIG. 19 depicts the binding epitope of Mab 228B/C-1. The human (SEQ IDNO: 187), monkey (SEQ ID NO: 188) and mouse (SEQ ID NO: 192) IL-13 aminoacid sequences are depicted.

FIG. 20 depicts the CDR variants and their respective SEQ ID Nos.

FIGS. 21A and 21B depict the variable light chain and variable heavychain sequences for select candidate recombinant antibodies.

DETAILED DESCRIPTION

This invention is not limited to the particular methodology, protocols,cell lines, vectors, or reagents described herein because they may vary.Further, the terminology used herein is for the purpose of describingparticular embodiments only and is not intended to limit the scope ofthe present invention. As used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise, e.g., reference to “a host cell”includes a plurality of such host cells.

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, theexemplary methods, devices, and materials are described herein.

All patents and publications mentioned herein are incorporated herein byreference to the extent allowed by law for the purpose of describing anddisclosing the proteins, enzymes, vectors, host cells, and methodologiesreported therein that might be used with the present invention. However,nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

Immunogen

Recombinant IL13 was used to immunize mice to generate the hybridomasthat produce the monoclonal antibodies of the present invention.Recombinant IL13 is commercially available from a number of sources(see, e.g. R & D Systems, Minneapolis, Minn., PeproTech, Inc., NJ, andSanofi Bio-Industries, Inc., Tervose, Pa.). Alternatively, a gene or acDNA encoding IL13 may be cloned into a plasmid or other expressionvector and expressed in any of a number of expression systems accordingto methods well known to those of skill in the art. Methods of cloningand expressing IL13 and the nucleic acid sequence for IL13 are wellknown (see, for example, U.S. Pat. No. 5,652,123). Because of thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding IL13 polypeptides may be produced. One may vary the nucleotidesequence by selecting combinations based on possible codon choices.These combinations are made in accordance with the standard tripletgenetic code as applied to the nucleotide sequence that codes fornaturally occurring IL13 polypeptide and all such variations are to beconsidered. Any one of these polypeptides may be used in theimmunization of an animal to generate antibodies that bind to IL13.

The immunogen IL13 polypeptide may, when beneficial, be expressed as afusion protein that has the IL13 polypeptide attached to a fusionsegment. The fusion segment often aids in protein purification, e.g., bypermitting the fusion protein to be isolated and purified by affinitychromatography. Fusion proteins can be produced by culturing arecombinant cell transformed with a fusion nucleic acid sequence thatencodes a protein including the fusion segment attached to either thecarboxyl and/or amino terminal end of the protein. Fusion segments mayinclude, but are not limited to, immunoglobulin Fc regions,glutathione-S-transferase, β-galactosidase, a poly-histidine segmentcapable of binding to a divalent metal ion, and maltose binding protein.

Exemplary polypeptides comprise all or a portion of SEQ ID NO. 1 orvariants thereof, or SEQ ID NO. 2 wherein amino acid 13 is Xaa and maybe changed from the wt, e.g, glutamic acid to lysine.

A fusion protein comprising a mutant form of human IL13 was used togenerate the antibodies of the present invention. This mutant form ofIL13 contained a single mutation resulting in an inactive form of theprotein (Thompson et al., J. Biol. Chem. 274: 2994 (1999)). In order togenerate neutralizing antibodies with high affinity, the fusion proteincomprised the mutant IL13 protein fused to an immunoglobulin Fc,specifically IgG1, and was expressed in a mammalian cell line such thatthe recombinant protein was naturally glycosylated. The Fc portion ofthe fusion protein may have provided a conformational structure thatexposed a key epitope. The glycosylation may have increased theimmunogenicity of the epitope, allowing the generation of antibodies tothis particular epitope.

IL13 polypeptides expressed in E. coli lack glycosylation and thecommercially available antibodies tested were generated using thisprotein. We tested these antibodies, e.g., R&D Systems and Pharmingen,and found that antibodies generated with an immunogen produced in E.coli do not cross react with the epitope bound by the antibodies of thepresent invention.

Antibody Generation

The antibodies of the present invention may be generated by any suitablemethod known in the art. The antibodies of the present invention maycomprise polyclonal antibodies. Methods of preparing polyclonalantibodies are known to the skilled artisan (Harlow, et al., Antibodies:a Laboratory Manual, (Cold spring Harbor Laboratory Press, 2nd ed.(1988), which is hereby incorporated herein by reference in itsentirety).

For example, an immunogen as described above may be administered tovarious host animals including, but not limited to, rabbits, mice, rats,etc., to induce the production of sera containing polyclonal antibodiesspecific for the antigen. The administration of the immunogen may entailone or more injections of an immunizing agent and, if desired, anadjuvant. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, and include but are not limitedto, Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Additionalexamples of adjuvants which may be employed include the MPL-TDM adjuvant(monophosphoryl lipid A, synthetic trehalose dicorynomycolate).Immunization protocols are well known in the art in the art and may beperformed by any method that elicits an immune response in the animalhost chosen. Adjuvants are also well known in the art.

Typically, the immunogen (with or without adjuvant) is injected into themammal by multiple subcutaneous or intraperitoneal injections, orintramuscularly or through IV. The immunogen may include an IL13polypeptide, a fusion protein or variants thereof. Depending upon thenature of the polypeptides (i.e., percent hydrophobicity, percenthydrophilicity, stability, net charge, isoelectric point etc.), it maybe useful to conjugate the immunogen to a protein known to beimmunogenic in the mammal being immunized. Such conjugation includeseither chemical conjugation by derivatizing active chemical functionalgroups to both the immunogen and the immunogenic protein to beconjugated such that a covalent bond is formed, or throughfusion-protein based methodology, or other methods known to the skilledartisan. Examples of such immunogenic proteins include, but are notlimited to, keyhole limpet hemocyanin, ovalbumin, serum albumin, bovinethyroglobulin, soybean trypsin inhibitor, and promiscuous T helperpeptides. Various adjuvants may be used to increase the immunologicalresponse as described above.

The antibodies of the present invention comprise monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma technology, suchas those described by Kohler and Milstein, Nature, 256:495 (1975) andU.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: A LaboratoryManual, (Cold spring Harbor Laboratory Press, 2.sup.nd ed. (1988), byHammerling, et al., Monoclonal Antibodies and T-Cell Hybridomas(Elsevier, N.Y., (1981)), or other methods known to the artisan. Otherexamples of methods which may be employed for producing monoclonalantibodies include, but are not limited to, the human B-cell hybridomatechnique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al.,1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. The hybridoma producing the MAb of this invention may becultivated in vitro or in vivo.

Using typical hybridoma techniques, a host such as a mouse, a humanizedmouse, a mouse with a human immune system, hamster, rabbit, camel or anyother appropriate host animal, is typically immunized with an immunogento elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to IL13. Alternatively,lymphocytes may be immunized in vitro with the antigen.

Generally, in making antibody-producing hybridomas, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (coding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986), pp.59-103). Immortalized cell lines are usually transformed mammaliancells, particularly myeloma cells of rodent, bovine or human origin.Typically, a rat or mouse myeloma cell line is employed. The hybridomacells may be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), substances thatprevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines may also be used for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theIL13. The binding specificity of monoclonal antibodies produced by thehybridoma cells is determined by, e.g., immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoadsorbant assay (ELISA). Such techniques are known in the art andwithin the skill of the artisan. The binding affinity of the monoclonalantibody to IL13 can, for example, be determined by a Scatchard analysis(Munson et al., Anal. Biochem., 107:220 (1980)).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640. Themonoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium by conventional immunoglobulinpurification procedures such as, e.g., protein A-sepharose,hydroxyapatite chromatography, gel exclusion chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

A variety of methods exist in the art for the production of monoclonalantibodies and thus, the invention is not limited to their soleproduction in hydridomas. For example, the monoclonal antibodies may bemade by recombinant DNA methods, such as those described in U.S. Pat.No. 4,816,567. In this context, the term “monoclonal antibody” refers toan antibody derived from a single eukaryotic, phage, or prokaryoticclone. The DNA encoding the monoclonal antibodies of the invention canbe readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of murine antibodies, orsuch chains from human, humanized, or other sources). The hydridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransformed into host cells such as NS0 cells, Simian COS cells, Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. The DNA also may be modified,for example, by substituting the coding sequence for human heavy andlight chain constant domains in place of the homologous murine sequences(U.S. Pat. No. 4,816,567; Morrison et al, supra) or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain cross-linking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to preventcross-linking.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entirety.

Humanized antibodies are antibody molecules generated in a non-humanspecies that bind the desired antigen having one or more complementaritydetermining regions (CDRs) from the non-human species and framework (FR)regions from a human immunoglobulin molecule. Often, framework residuesin the human framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988),which are incorporated herein by reference in their entireties).Antibodies can be humanized using a variety of techniques known in theart including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chainshuffling (U.S. Pat. No. 5,565,332).

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the methods of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossible some FR residues are substituted from analogous sites in rodentantibodies.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety. The techniques of Cole et al., andBoerder et al., are also available for the preparation of humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol.,147(1):86-95, (1991)).

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc.(Princeton, N.J.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Also human MAbs could be made by immunizing mice transplanted with humanperipheral blood leukocytes, splenocytes or bone marrows (e.g., Triomatechniques of XTL). Completely human antibodies which recognize aselected epitope can be generated using a technique referred to as“guided selection.” In this approach a selected non-human monoclonalantibody, e.g., a mouse antibody, is used to guide the selection of acompletely human antibody recognizing the same epitope. (Jespers et al.,Bio/technology 12:899-903 (1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

The antibodies of the present invention may be bispecific antibodies.Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present invention, one of the binding specificities maybe directed towards IL13, the other may be for any other antigen, andpreferably for a cell-surface protein, receptor, receptor subunit,tissue-specific antigen, virally derived protein, virally encodedenvelope protein, bacterially derived protein, or bacterial surfaceprotein, etc.

Methods for making bispecific antibodies are well known. Traditionally,the recombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy-chain/light-chain pairs, wherethe two heavy chains have different specificities (Milstein and Cuello,Nature, 305:537-539 (1983). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published May13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It may have the first heavy-chainconstant region (CH1) containing the site necessary for light-chainbinding present in at least one of the fusions. DNAs encoding theimmunoglobulin heavy-chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transformed into a suitable host organism. For further details ofgenerating bispecific antibodies see, for example Suresh et al., Meth.In Enzym., 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980).It is contemplated that the antibodies may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioester bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980.

In addition, one can generate single-domain antibodies to IL-13.Examples of this technology have been described in WO9425591 forantibodies derived from Camelidae heavy chain Ig, as well inUS20030130496 describing the isolation of single domain fully humanantibodies from phage libraries.

Identification of Anti-IL13 Antibodies

The present invention provides antagonist monoclonal antibodies thatinhibit and neutralize the action of IL13. In particular, the antibodiesof the present invention bind to IL13 and inhibit the activation of theIL13 receptor alpha chain-1 (IL13Rα1). The antibodies of the presentinvention include the antibodies designated 228B/C-1, 228A-4, 227-26,and 227-43, and humanized clones of 228B/C-1 are disclosed. The presentinvention also includes antibodies that bind to the same epitope as oneof these antibodies, e.g., that of monoclonal antibody 228B/C-1.

Candidate anti-IL13 antibodies were tested by enzyme linkedimmunosorbent assay (ELISA), Western immunoblotting, or otherimmunochemical techniques. Assays performed to characterize theindividual antibodies included: (1) Inhibition of IL13-autocrineproliferation of Hodgkin's lymphoma cell lines HDLM-2 and L-1236; (2)Inhibition of IL13-induced STAT6 phosphorylation in THP-1 cells; and (3)Inhibition of IL13-induced suppression of CD14 expression in primaryhuman monocytes; and (4) Inhibition of IL13-induced up-regulation ofCD23 expression on primary human monocytes. Experimental details aredescribed in the Examples.

Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, monovalent, bispecific, heteroconjugate, multispecific,human, humanized or chimeric antibodies, single chain antibodies,single-domain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above.

The term “antibody,” as used herein, refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.Moreover, the term “antibody” (Ab) or “monoclonal antibody” (MAb) ismeant to include intact molecules, as well as, antibody fragments (suchas, for example, Fab and F(ab′)₂ fragments) which are capable ofspecifically binding to a protein. Fab and F(ab′)₂ fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation ofthe animal or plant, and may have less non-specific tissue binding thanan intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

The antibodies may be human antigen-binding antibody fragments of thepresent invention and include, but are not limited to, Fab, Fab′ andF(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and single-domain antibodies comprisingeither a VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are from human, non-human primates,rodents (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig,camel, horse, or chicken.

As used herein, “human” antibodies” include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of IL13 or may be specific for bothIL13 as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of IL13 which they recognize orspecifically bind. The epitope(s) or polypeptide portion(s) may bespecified as described herein, e.g., by N-terminal and C-terminalpositions, by size in contiguous amino acid residues, or listed in theTables and Figures.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that bind IL13polypeptides, which have at least 95%, at least 90%, at least 85%, atleast 80%, at least 75%, at least 70%, at least 65%, at least 60%, atleast 55%, and at least 50% identity (as calculated using methods knownin the art and described herein) to IL-13 are also included in thepresent invention. Anti-IL-13 antibodies may also bind with a K_(D) ofless than about 10⁻⁷ M, less than about 10⁻⁶ M, or less than about 10⁻⁵M to other proteins, such as IL-13 antibodies from species other thanthat against which the anti-IL-13 antibody is directed.

In specific embodiments, antibodies of the present invention cross-reactwith monkey homologues of human IL13 and the corresponding epitopesthereof. In a specific embodiment, the above-described cross-reactivityis with respect to any single specific antigenic or immunogenicpolypeptide, or combination(s) of the specific antigenic and/orimmunogenic polypeptides disclosed herein.

Further included in the present invention are antibodies which bindpolypeptides encoded by polynucleotides which hybridize to apolynucleotide encoding IL13 under stringent hybridization conditions.Antibodies of the present invention may also be described or specifiedin terms of their binding affinity to a polypeptide of the invention.Preferred binding affinities include those with an equilibriumdissociation constant or K_(D) from 10⁻⁸ to 10⁻¹⁵ M, 10⁻⁸ to 10⁻¹² M,10⁻⁸ to 10⁻¹⁰ M, or 10⁻¹⁰ to 10⁻¹² M. The invention also providesantibodies that competitively inhibit binding of an antibody to anepitope of the invention as determined by any method known in the artfor determining competitive binding, for example, the immunoassaysdescribed herein. In preferred embodiments, the antibody competitivelyinhibits binding to the epitope by at least 95%, at least 90%, at least85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least50%.

Vectors and Host Cells

In another aspect, the present invention provides vector constructscomprising a nucleotide sequence encoding the antibodies of the presentinvention and a host cell comprising such a vector. Standard techniquesfor cloning and transformation may be used in the preparation of celllines expressing the antibodies of the present invention.

Recombinant expression vectors containing a nucleotide sequence encodingthe antibodies of the present invention can be prepared using well knowntechniques. The expression vectors include a nucleotide sequenceoperably linked to suitable transcriptional or translational regulatorynucleotide sequences such as those derived from mammalian, microbial,viral, or insect genes. Examples of regulatory sequences includetranscriptional promoters, operators, enhancers, mRNA ribosomal bindingsites, and/or other appropriate sequences which control transcriptionand translation initiation and termination. Nucleotide sequences are“operably linked” when the regulatory sequence functionally relates tothe nucleotide sequence for the appropriate polypeptide. Thus, apromoter nucleotide sequence is operably linked to, e.g., the antibodyheavy chain sequence if the promoter nucleotide sequence controls thetranscription of the appropriate nucleotide sequence.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with antibody heavy and/or light chain sequencescan be incorporated into expression vectors. For example, a nucleotidesequence for a signal peptide (secretory leader) may be fused in-frameto the polypeptide sequence so that the antibody is secreted to theperiplasmic space or into the medium. A signal peptide that isfunctional in the intended host cells enhances extracellular secretionof the appropriate antibody. The signal peptide may be cleaved from thepolypeptide upon secretion of antibody from the cell. Examples of suchsecretory signals are well known and include, e.g., those described inU.S. Pat. No. 5,698,435, U.S. Pat. No. 5,698,417, and U.S. Pat. No.6,204,023.

Host cells useful in the present invention include but are not limitedto microorganisms such as bacteria (e.g., E. coli, B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,Baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 3T3 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter).

The vector may be a plasmid vector, a single or double-stranded phagevector, or a single or double-stranded RNA or DNA viral vector. Suchvectors may be introduced into cells as polynucleotides by well knowntechniques for introducing DNA and RNA into cells. The vectors, in thecase of phage and viral vectors also may be introduced into cells aspackaged or encapsulated virus by well known techniques for infectionand transduction. Viral vectors may be replication competent orreplication defective. In the latter case, viral propagation generallywill occur only in complementing host cells. Cell-free translationsystems may also be employed to produce the protein using RNAs derivedfrom the present DNA constructs. Such vectors may include the nucleotidesequence encoding the constant region of the antibody molecule (see,e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S.Pat. No. 5,122,464) and the variable domain of the antibody may becloned into such a vector for expression of the entire heavy or lightchain.

Prokaryotes useful as host cells in the present invention include gramnegative or gram positive organisms such as E. coli, and B. subtilis.Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec,Madison, Wis., USA), and the pET (Novagen, Madison, Wis., USA) and pRSET(Invitrogen Corporation, Carlsbad, Calif., USA) series of vectors(Studier, F. W., J. Mol. Biol. 219: 37 (1991); Schoepfer, R. Gene 124:83 (1993)). Promoter sequences commonly used for recombinant prokaryotichost cell expression vectors include T7, (Rosenberg, et al. Gene 56,125-135 (1987)), β-lactamase (penicillinase), lactose promoter system(Chang et al., Nature 275:615, (1978); and Goeddel et al., Nature281:544, (1979)), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, (1980)), and tac promoter (Sambrook et al.,1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.)

Yeasts useful in the present invention include those from the genusSaccharomyces, Pichia, Actinomycetes and Kluyveromyces. Yeast vectorswill often contain an origin of replication sequence from a 2μ yeastplasmid, an autonomously replicating sequence (ARS), a promoter region,sequences for polyadenylation, sequences for transcription termination,and a selectable marker gene. Suitable promoter sequences for yeastvectors include, among others, promoters for metallothionein,3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073,(1980)) or other glycolytic enzymes (Holland et al., Biochem. 17:4900,(1978)) such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Fleer et al., Gene, 107:285-195(1991). Other suitable promoters and vectors for yeast and yeasttransformation protocols are well known in the art. Yeast transformationprotocols are well known. One such protocol is described by Hinnen etal., Proc. Natl. Acad. Sci., 75:1929 (1978). The Hinnen protocol selectsfor Trp⁺ transformants in a selective medium.

Mammalian or insect host cell culture systems may also be employed toexpress recombinant antibodies, e.g., Baculovirus systems for productionof heterologous proteins. In an insect system, Autographa californicanuclear polyhedrosis virus (AcNPV) may be used as a vector to expressforeign genes. The virus grows in Spodoptera frugiperda cells. Theantibody coding sequence may be cloned individually into non-essentialregions (for example the polyhedrin gene) of the virus and placed undercontrol of an AcNPV promoter (for example the polyhedrin promoter).

NS0 or Chinese hamster ovary (CHO) cells for mammalian expression of theantibodies of the present invention may be used. Transcriptional andtranslational control sequences for mammalian host cell expressionvectors may be excised from viral genomes. Commonly used promotersequences and enhancer sequences are derived from Polyoma virus,Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus (CMV).DNA sequences derived from the SV40 viral genome may be used to provideother genetic elements for expression of a structural gene sequence in amammalian host cell, e.g., SV40 origin, early and late promoter,enhancer, splice, and polyadenylation sites. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication. Exemplary expression vectors for use in mammalian hostcells are commercially available.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides or nucleic acids, e.g.,DNA, comprising a nucleotide sequence encoding an antibody of theinvention and fragments thereof. Exemplary polynucleotides include thoseencoding antibody chains comprising one or more of the amino acidsequences described herein. The invention also encompassespolynucleotides that hybridize under stringent or lower stringencyhybridization conditions to polynucleotides that encode an antibody ofthe present invention.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A⁺ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the CDRs by well known methods, e.g., by comparison to known aminoacid sequences of other heavy and light chain variable regions todetermine the regions of sequence hypervariability. Using routinerecombinant DNA techniques, one or more of the CDRs may be insertedwithin framework regions, e.g., into human framework regions to humanizea non-human antibody, as described supra. The framework regions may benaturally occurring or consensus framework regions, and preferably humanframework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479(1998) for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine MAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing Anti-IL13 Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody or a fragment of the antibody.Once a polynucleotide encoding an antibody molecule has been obtained,the vector for the production of the antibody may be produced byrecombinant DNA technology. An expression vector is constructedcontaining antibody coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. In one aspect of theinvention, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention as described above. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. Bacterial cells such as E. coli, and eukaryoticcells are commonly used for the expression of a recombinant antibodymolecule, especially for the expression of whole recombinant antibodymolecule. For example, mammalian cells such as Chinese hamster ovarycells (CHO), in conjunction with a vector such as the major intermediateearly gene promoter element from human cytomegalovirus is an effectiveexpression system for antibodies (Foecking et al., Gene 45:101 (1986);Cockett et al., Bio/Technology 8:2 (1990)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include, but are not limited to, CHO, COS, 293, 3T3, or myelomacells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk, hgprt or aprt− cells, respectively. Also, antimetaboliteresistance can be used as the basis of selection for the followinggenes: dhfr, which confers resistance to methotrexate (Wigler et al.,Proc. Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418 (Wuand Wu, Biotherapy 3:87-95 (1991)); and hygro, which confers resistanceto hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonlyknown in the art of recombinant DNA technology may be routinely appliedto select the desired recombinant clone, and such methods are described,for example, in Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer andExpression, A Laboratory Manual, Stockton Press, NY (1990); and inChapters 12 and 13, Dracopoli et al. (eds), Current Protocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.Biol. 150:1 (1981), which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, “The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells” (DNA Cloning, Vol. 3. Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and size-exclusion chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide. Fused or conjugated antibodies of thepresent invention may be used for ease in purification. See e.g., Harboret al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., Proc. Natl. Acad. Sci. 89:1428-1432 (1992); Fell et al., J.Immunol. 146:2446-2452 (1991), which are incorporated by reference intheir entireties.

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

Diagnostic Uses for Anti-IL13 Antibodies

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to theantibody, such that covalent attachment does not interfere with bindingto IL13. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., bybiotinylation, HRP, or any other detectable moiety.

Antibodies of the present invention may be used, for example, but notlimited to, to purify or detect IL13, including both in vitro and invivo diagnostic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofIL13 in biological samples. See, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988)(incorporated by reference herein in its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic agent. The antibodies can be useddiagnostically to, for example, monitor the development or progressionof an allergic response as part of a clinical testing procedure to,e.g., determine the efficacy of a given treatment regimen. Detection canbe facilitated by coupling the antibody to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, radioactive materials, positron emitting metals using variouspositron emission tomographies, and nonradioactive paramagnetic metalions. The detectable substance may be coupled or conjugated eitherdirectly to the antibody (or fragment thereof) or indirectly, through anintermediate (such as, for example, a linker known in the art) usingtechniques known in the art. See, for example, U.S. Pat. No. 4,741,900for metal ions which can be conjugated to antibodies for use asdiagnostics according to the present invention. Examples of suitableenzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ¹¹¹Inor ⁹⁹Tc.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to IL13 can be used for diagnostic purposes to detect,diagnose, or monitor diseases, disorders, and/or conditions associatedwith the aberrant expression and/or activity of IL13. The inventionprovides for the detection of aberrant expression of IL13, comprising(a) assaying the expression of IL13 in cells or body fluid of anindividual using one or more antibodies of the present inventionspecific to IL13 and (b) comparing the level of gene expression with astandard gene expression level, whereby an increase or decrease in theassayed IL13 expression level compared to the standard expression levelis indicative of aberrant expression.

Antibodies may be used for detecting the presence and/or levels of IL13in a sample, e.g., a bodily fluid or tissue sample. The detecting methodmay comprise contacting the sample with an IL13 antibody and determiningthe amount of antibody that is bound to the sample.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of IL13 in cells or body fluid ofan individual using one or more antibodies of the present invention and(b) comparing the level of gene expression with a standard geneexpression level, whereby an increase or decrease in the assayed geneexpression level compared to the standard expression level is indicativeof a particular disorder.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (⁹⁹Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of IL13 in an animal,preferably a mammal and most preferably a human. In one embodiment,diagnosis comprises: a) administering (for example, parenterally,subcutaneously, or intraperitoneally) to a subject an effective amountof a labeled molecule which specifically binds to IL13; b) waiting for atime interval following the administration permitting the labeledmolecule to preferentially concentrate at sites in the subject where thepolypeptide is expressed (and for unbound labeled molecule to be clearedto background level); c) determining background level; and d) detectingthe labeled molecule in the subject, such that detection of labeledmolecule above the background level indicates that the subject has aparticular disease or disorder associated with aberrant expression ofIL13. Background level can be determined by various methods including,comparing the amount of labeled molecule detected to a standard valuepreviously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ⁹⁹ Tc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo imaging is describedin S. W. Burchiel et al., “Immunopharmacokinetics of RadiolabeledAntibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: TheRadiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes,eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

In another aspect, the present invention provides a method fordiagnosing the predisposition of a patient to develop diseases caused bythe unregulated expression of cytokines. Increased amounts of IL13 incertain patient cells, tissues, or body fluids may indicate that thepatient is predisposed to certain immune diseases. In one embodiment,the method comprises collecting a cell, tissue, or body fluid sample asubject known to have low or normal levels of IL13, analyzing the tissueor body fluid for the presence of IL13 in the tissue, and predicting thepredisposition of the patient to certain immune diseases based upon thelevel of expression of IL13 in the tissue or body fluid. In anotherembodiment, the method comprises collecting a cell, tissue, or bodyfluid sample known to contain a defined level of IL13 from a patient,analyzing the tissue or body fluid for the amount of IL13, andpredicting the predisposition of the patient to certain immune diseasesbased upon the change in the amount of IL13 compared to a defined ortested level established for normal cell, tissue, or bodily fluid. Thedefined level of IL13 may be a known amount based upon literature valuesor may be determined in advance by measuring the amount in normal cell,tissue, or body fluids. Specifically, determination of IL13 levels incertain tissues or body fluids permits specific and early, preferablybefore disease occurs, detection of immune diseases in the patient.Immune diseases that can be diagnosed using the present method include,but are not limited to, the immune diseases described herein. In thepreferred embodiment, the tissue or body fluid is peripheral blood,peripheral blood leukocytes, biopsy tissues such as lung or skinbiopsies, and tissue.

Therapeutic Uses of Anti-IL13 Antibodies

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) can beused as a therapeutic. The present invention is directed toantibody-based therapies which involve administering antibodies of theinvention to an animal, a mammal, or a human, for treating anIL13-mediated disease, disorder, or condition. The animal or subject maybe an animal in need of a particular treatment, such as an animal havingbeen diagnosed with a particular disorder, e.g., one relating to IL13.Antibodies directed against IL13 are useful for inhibiting allergicreactions in animals, including but not limited to cows, pigs, horses,chickens, cats, dogs, non-human primates etc., as well as humans. Forexample, by administering a therapeutically acceptable dose of anantibody, or antibodies, of the present invention, or a cocktail of thepresent antibodies, or in combination with other antibodies of varyingsources, an allergic response to antigens may be reduced or eliminatedin the treated mammal.

Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention as described below (including fragments,analogs and derivatives thereof and anti-idiotypic antibodies asdescribed herein). The antibodies of the invention can be used to treat,inhibit or prevent diseases, disorders or conditions associated withaberrant expression and/or activity of 103, including, but not limitedto, any one or more of the diseases, disorders, or conditions describedherein. The treatment and/or prevention of diseases, disorders, orconditions associated with aberrant expression and/or activity of IL13includes, but is not limited to, alleviating at least one symptomsassociated with those diseases, disorders or conditions. Antibodies ofthe invention may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein.

Anti-IL13 antibodies of the present invention may be usedtherapeutically in a variety of diseases. The present invention providesa method for preventing or treating IL13-mediated diseases in a mammal.The method comprises administering a disease preventing or treatingamount of anti-IL13 antibody to the mammal. The anti-IL13 antibody bindsto IL13 and regulates cytokine and cellular receptor expressionresulting in cytokine levels characteristic of non-disease states. Thus,diseases for treatment include allergy, asthma, autoimmune disease, orother inflammatory diseases. Other allergic diseases include allergicrhinitis, atopic dermatitis, food hypersensitivity and urticaria;immune-mediated skin diseases include bullous skin diseases, erythemamultiform and contact dermatitis; autoimmune disease include psoriasis,rheumatoid arthritis, juvenile chronic arthritis; inflammatory boweldisease (i.e., ulcerative colitis, Crohn's disease); other diseasesassociated with IL13 include idiopathic interstitial pneumonia, gobletcell metaplasia, inflammatory and fibrotic lung diseases such as cysticfibrosis, gluten-sensitive enteropathy, and Whipple's disease;immunologic diseases of the lung such as eosinophilic pneumonia,idiopathic pulmonary fibrosis and hypersensitivity pneumonitis; chronicobstructive pulmonary disease, RSV infection, uvelitis, scleroderma,osteoporosis, and Hodgkin's lymphoma.

The amount of the antibody which will be effective in the treatment,inhibition and prevention of a disease or disorder associated withaberrant expression and/or activity of IL13 can be determined bystandard clinical techniques. The antibody can be administered intreatment regimes consistent with the disease, e.g., a single or a fewdoses over one to several days to ameliorate a disease state or periodicdoses over an extended time to prevent allergy or asthma. In addition,in vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patients body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatients body weight, more preferably 1 mg/kg to 10 mg/kg of thepatients body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3IL-7, IFN), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments, such as immunotherapy,bronchodilators, anti-IgE molecules, anti-histamines, oranti-leukotrienes.

In a preferred aspect, the antibody is substantially purified (e.g.,substantially free from substances that limit its effect or produceundesired side-effects).

Various delivery systems are known and can be used to administer anantibody of the present invention, including injection, e.g.,encapsulation in liposomes, microparticles, microcapsules, recombinantcells capable of expressing the compound, receptor-mediated endocytosis(see, e.g., Wu et al., J. Biol. Chem. 262:4429-4432 (1987)),construction of a nucleic acid as part of a retroviral or other vector,etc.

The anti-IL13 antibody can be administered to the mammal in anyacceptable manner. Methods of introduction include but are not limitedto intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, inhalation and oral routes. Theantibodies or compositions may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the therapeutic antibodies orcompositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. Theantibody may also be administered into the lungs of a patient in theform of a dry powder composition (See e.g., U.S. Pat. No. 6,514,496).

In a specific embodiment, it may be desirable to administer thetherapeutic antibodies or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion, topical application, by injection,by means of a catheter, by means of a suppository, or by means of animplant, said implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.Preferably, when administering an antibody of the invention, care mustbe taken to use materials to which the protein does not absorb.

In another embodiment, the antibody can be delivered in a vesicle, inparticular a liposome (see Langer, Science 249:1527-1533 (1990); Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In yet another embodiment, the antibody can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989)). In another embodiment, polymeric materials can be used (seeMedical Applications of Controlled Release, Langer and Wise (eds.), CRCPres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley, New York(1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of theantibody, and a physiologically acceptable carrier. In a specificembodiment, the term “physiologically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such physiological carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable carriers are described in “Remington's Pharmaceutical Sciences”by E. W. Martin. Such compositions will contain an effective amount ofthe antibody, preferably in purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. The formulation should suit the mode of administration.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

In addition, the antibodies of the present invention may be conjugatedto various effector molecules such as heterologous polypeptides, drugs,radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387. Anantibody or fragment thereof may be conjugated to a therapeutic moietysuch as a cytotoxin, e.g., a cytostatic or cytocidal agent, atherapeutic agent or a radioactive metal ion, e.g., alpha-emitters suchas, for example, 213Bi. A cytotoxin or cytotoxic agent includes anyagent that is detrimental to cells. Examples include paclitaxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologues thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate.(See, e.g., Segal in U.S. Pat. No. 4,676,980.)

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-α, TNF-β, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibody-Based Gene Therapy

In a another aspect of the invention, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of IL13, by way of gene therapy.Gene therapy refers to therapy performed by the administration to asubject of an expressed or expressible nucleic acid. In this embodimentof the invention, the nucleic acids produce their encoded protein thatmediates a therapeutic effect. Any of the methods for gene therapyavailable can be used according to the present invention. Exemplarymethods are described below.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993).

In a one aspect, the compound comprises nucleic acid sequences encodingan antibody, said nucleic acid sequences being part of expressionvectors that express the antibody or fragments or chimeric proteins orheavy or light chains thereof in a suitable host. In particular, suchnucleic acid sequences have promoters operably linked to the antibodycoding region, said promoter being inducible or constitutive, and,optionally, tissue-specific.

In another particular embodiment, nucleic acid molecules are used inwhich the antibody coding sequences and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe antibody encoding nucleic acids (Koller and Smithies, Proc. Natl.Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438(1989). In specific embodiments, the expressed antibody molecule is asingle chain antibody; alternatively, the nucleic acid sequences includesequences encoding both the heavy and light chains, or fragmentsthereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates the delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdrl gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. Gen. and Dev. 3:110-114 (1993).

Adenoviruses may also be used in the present invention. Adenoviruses areespecially attractive vehicles in the present invention for deliveringantibodies to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia. Other targets for adenovirus-based deliverysystems are liver, the central nervous system, endothelial cells, andmuscle. Adenoviruses have the advantage of being capable of infectingnon-dividing cells. Kozarsky and Wilson, Curr. Opin. Gen. Dev. 3:499-503(1993) present a review of adenovirus-based gene therapy. Bout et al.,Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirusvectors to transfer genes to the respiratory epithelia of rhesusmonkeys. Other instances of the use of adenoviruses in gene therapy canbe found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld etal., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest.91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., GeneTherapy 2:775-783 (1995). Adeno-associated virus (AAV) has also beenproposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol.Med. 204:289-300 (1993); U.S. Pat. Nos. 5,436,146; 6,632,670;6,642,051).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a one embodiment, the cell used for gene therapy is autologous to thepatient. Nucleic acid sequences encoding an antibody of the presentinvention are introduced into the cells such that they are expressibleby the cells or their progeny, and the recombinant cells are thenadministered in vivo for therapeutic effect. In a specific embodiment,stem or progenitor cells are used. Any stem and/or progenitor cellswhich can be isolated and maintained in vitro can potentially be used inaccordance with this embodiment of the present invention (see e.g. PCTPublication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992);Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, MayoClinic Proc. 61:771 (1986)).

EXAMPLES Example 1 Preparation of IL13 Immunogen: A Mutated, InactiveHuman IL13/Fc (MT-IL13/Fc)

A. Cloning and Construction of an Expression Plasmid for MT-IL13/Fc

It was reported that human IL13 with a mutation (glutamic acid tolysine) at amino acid residue #13 bound IL13Rα1 with equal or higheraffinity but had lost the ability to activate IL13Rα1-bearing cells(Thompson et al., J. Biol. Chem., 274: 29944 (1999)). This mutated,inactive IL13, designated MT-IL13, was expressed in human embryonickidney cells 293-T. The purified recombinant protein was used as theimmunogen in the present invention to generate anti-IL13 monoclonalantibodies. Two oligonucleotide primers:

(SEQ ID NO 9) 5′ AAGCTTTCCCCAGGCCCTGTGCCTCCCTCTACAGCCCTCAGGAAGCT CAT3′(SEQ ID NO 10) 5′ CTCGAGGTTGAACCGTCCCTCGCGAAAAAG 3′

corresponding to the oligonucleotide sequence of MT-IL13 gene weresynthesized and used as templates in polymerase chain reactions (PCR) toclone the IL13 gene from human testis cDNA library (BD BiosciencesClontech, Palo Alto, Calif.). The PCR fragment (342 base pairs) whichlacked the predicted signal peptide sequence of IL13 was ligated intothe pSecTag/FRT vector (Invitrogen, Carlsbad, Calif.) that contained asecretion signal peptide sequence at the 5′ end and a human Fcγ1 (hingeand constant regions CH2 and CH3) sequence at the 3′ end. Theconstruct's composition was confirmed by sequencing.

B. Production of MT-IL13/Fc from Transfected 293T Cells

For transient expression of MT-IL13/Fc, purified plasmid DNA wastransfected into 293T cells by Lipofectamine 2000 (Invitrogen),according to the manufacturer's protocol. At 72 hours post-transfection,culture supernatants from transfected cells were collected forpurification. For stable expression of MT-IL13/Fc, cell lines wereestablished using a Flp-In 293T cell line (Invitrogen). To confirmexpression, culture supernatants were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The separated proteinswere transferred to nitrocellulose membrane and detected by reactionwith horseradish peroxidase (HRP) conjugated mouse anti-human IgG (Fc)monoclonal antibody (Sigma, St. Louis, Mo.) or polyclonal goat anti-IL13antibodies (R&D Systems, Minneapolis, Minn.), which were then detectedwith HRP-donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, WestGrove, Pa.). The immunoreactive proteins were identified on film, usingenhanced chemi-luminescence detection (Supersignal West PicoChemiluminescent Substrate, Pierce, Rockford, Ill.).

C. Purification of MTIL13/Fc

MT-IL13/Fc was purified with a hyper-D protein A affinity column(Invitrogen) equilibrated with phosphate-buffered saline (PBS). Afterapplying the cell culture supernatant to the column, the resin waswashed with more than 20 column volumes of PBS. Then, the resin waswashed with SCC buffer (0.05 M sodium citrate, 0.5 M sodium chloride, pH6.0) to remove unbound proteins. The IL13 fusion proteins were theneluted (0.05 M sodium citrate, 0.15 M sodium chloride, pH 3.0) anddialyzed in PBS.

Fractions from the affinity column containing MT-IL13/Fc were analyzedby SDS-PAGE. The purity of the proteins were analyzed by Coomassie Bluestaining and the identity of the proteins by Western immunoblottingusing goat anti-human IgG (Fc) antibody (Sigma) and goat anti-human IL13antibody (R&D Systems) as described above.

Example 2 Generation of Anti-IL13 Monoclonal Antibodies

Male A/J mice (Harlan, Indianapolis, Ind.), 8-12 weeks old, wereinjected subcutaneously with 20 μg MT-IL13/Fc in complete Freund'sadjuvant (Difco Laboratories, Detroit, Mich.) in 200 μL of PBS pH 7.4.At two-week intervals the mice were twice injected subcutaneously with20 μg MT-IL13/Fc in incomplete Freund's adjuvant. Then, two weeks laterand three days prior to sacrifice, the mice were again injectedintraperitoneally with 20 μg of the same immunogen in PBS. Spleen cellsisolated from one or more antigen-immunized mouse were used for fusion.Similar procedures of immunization and fusion were also used with E.coli expressed human IL13 (R&D Systems) as immunogen.

In the fusion leading to the generation of the anti-IL13 MAb 228B/C-1,26.4×10⁶ spleen cells and 58.8×10⁶ spleen cells from two immunized micewere combined. For each fusion, single cell suspensions were preparedfrom the spleen of immunized mice and used for fusion with Sp2/0 myelomacells. Sp2/0 and spleen cells at a ratio of 1:1 were fused in a mediumcontaining 50% polyethylene glycol (M.W. 1450) (Kodak, Rochester, N.Y.)and 5% dimethylsulfoxide (Sigma). The cells were then adjusted to aconcentration of 1.5×10⁵ spleen cells per 250 μL of the suspension inDMEM medium (Invitrogen, CA), supplemented with 10% fetal bovine serum,100 units/mL of penicillin, 100 μg/mL of streptomycin, 0.1 mMhypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine. Two hundred andfifty microliters of the cell suspension were added to each well ofabout fifty 96-well microculture plates. After about ten days culturesupernatants were withdrawn for screening for reactivity with MT-IL13/Fcin ELISA.

Wells of Immulon 2 (Dynatech Laboratories, Chantilly, Va.) microtestplates were coated by adding purified MT-IL13/Fc (0.1 μg/mL) overnightat room temperature. After the coating solution was removed by flickingof the plate, 200 μL of a blocking/diluting buffer (PBS containing 2%bovine serum albumin and 0.05% TWEEN® 20) was added to each well for onehour to block the non-specific sites. One hour later, the wells werethen washed with PBST buffer (PBS containing 0.05% TWEEN® 20). Fiftymicroliters of culture supernatant was collected from each fusion well,mixed with 50 μL of the blocking/diluting buffer and then added to theindividual wells of the microtest plates. After one hour of incubation,the wells were washed with PBST. The bound murine antibodies were thendetected by reaction with HRP-conjugated goat anti-mouse IgG (Fcspecific) (Jackson ImmunoResearch Lab, West Grove, Pa.) and diluted at1:2,000 with the blocking/diluting buffer. Peroxidase substrate solutioncontaining 0.1% 3,3,5,5 tetramethyl benzidine (Sigma, St. Louis, Mo.)and 0.003% hydrogen peroxide (Sigma) was added to the wells for colordevelopment for 30 minutes. The reaction was terminated by the additionof 50 μL of 2 M H₂SO₄ per well. The OD₄₅₀ of the reaction mixture wasmeasured with a BioTek ELISA Reader (BioTek Instruments, Winooski, Vt.).

The culture supernatants from the positive wells of MT-IL13/Fc screeningwere then tested for negative binding to an irrelevant Fγ1 fusionprotein. Final positive wells were then selected for single-cell cloningby limiting dilution. Culture supernatants from monoclonal antibodieswere re-tested to confirm their reactivity by ELISA. Selected hybridomaswere grown in spinner flasks and the spent culture supernatant collectedfor antibody purification by protein A affinity chromatography.

The purified antibodies were tested by four assays: i) Cross-reactivitywith 293T cell expressed MT-IL13/Fc and E. coli expressed mouse IL13;ii) Inhibition of IL13-autocrine proliferation of HDLM-2 and L-1236cells; iii) Inhibition of IL13-induced STATE phosphorylation in THP-1cells; and iv) Inhibition of IL13-regulated CD14 and CD23 expression onhuman monocytes.

Seventy-three anti-IL13 MAbs were obtained from the fusions performed onMT-IL13/Fc and IL13 immunized mice. Thirty-nine of these MAbs werepurified for characterization by ELISA and cell-based assays. Thirteenof these 39 MAbs inhibited autocrine IL13-induced proliferation ofHDLM-2 and L-1236 cells (see assay description and results in Example5). Four of the MAbs were found to be very strongly reactive with humanIL13 in ELISA and were neutralizing against human IL13 in functionalcell-based assays. These MAbs were designated 228B/C-1, 228A-4, 227-26,and 227-43. These antibodies were all generated using the glycosylatedMT-IL13/Fc as immunogen.

Example 3 Reactivity of Anti-IL13 Monoclonal Antibodies with Human andMouse IL13 in ELISA

The reactivity of various anti-IL13 monoclonal antibodies was tested byELISA. Different wells of 96-well microtest plates were coated witheither E. coli expressed non-glycosylated human IL13 (R&D Systems), 293Tcell expressed glycosylated MT-IL13/Fc, or E. coli expressed mouse IL13(R&D Systems) by the addition of 100 μL of IL13 protein at 0.1 μg/mL inPBS. After overnight incubation at room temperature, the wells weretreated with PBSTB (PBST containing 2% BSA) to saturate the remainingbinding sites. The wells were then washed with PBST.

One hundred microliters of two-fold serially diluted anti-IL13 MAbs (0.5μg/mL (3.33 nM) to 0.05 ng/mL (0.00033 nM)) were added to the wells for1 hour at room temperature. An anti-IL13 MAb JES-5A2 from (BDBiosciences-Pharmingen, San Diego, Calif.) was also tested as a positivecontrol. This antibody was generated by using E. coli expressed humanIL13 as immunogen. An isotype-matched mouse anti-HIV-1 gp120 MAb wasused as irrelevant negative control. The wells were then washed withPBST. Bound antibody was detected by incubation with diluted HRP-goatanti-mouse IgG (Fc) (Jackson ImmunoResearch) for 1 hour at roomtemperature. Peroxidase substrate solution was then added for colordevelopment as described above. The OD₄₅₀ was measured using an ELISAreader.

FIG. 1 shows the dose-dependent binding of anti-IL13 MAbs 228B/C-1,228A-4, 227-26, 227-43, and the negative control in ELISA. Among theseMAbs, 228B/C-1 showed the strongest reactivity. FIG. 2 shows thedose-dependent binding of the anti-IL13 MAbs to MT-IL13/Fc in ELISA.228B/C-1 and 228A-4 showed the strongest reactivity with MT-IL13/Fc,whereas 227-26 and 227-43 showed moderate reactivity.

FIGS. 1 and 2 show that 228B/C-1 has highest affinity for bothglycosylated and non-glycosylated human IL13 among all the anti-IL13MAbs tested. All these anti-IL13 MAbs did not cross-react with mouseIL13 in ELISA (data no shown).

Example 4 Lack of Competition of 228B/C-1-Hrp Binding to Human IL13 byJES10-5A2

To address whether JES10-5A2 and 228B/C-1 bind to the same epitope onhuman IL13, a competition ELISA was used to examine the effect ofJES10-5A2 on 228B/C-1-HRP binding to E. coli expressed human IL13. Eachwell of 96-well microtest plates were incubated with 100 μL of IL13protein at 0.1 μg/mL in PBS. After overnight incubation at roomtemperature, the wells were treated with PBSTB (PBST containing 2% BSA)to saturate the remaining binding sites. The wells were then washed withPBST. Fifty microliters of two fold serially diluted 228B/C-1 andJES10-5A2 (from a final concentration of 20 μg/mL to 9.76 ng/mL) weremixed with 50 μL of pre-titrated 228B/C-1-HRP (at 1:6,400 dilution). Themixtures were then added to the wells and incubated for 1 hour at roomtemperature. Peroxidase substrate solution was then added for colordevelopment as described above. The OD₄₅₀ was measured using an ELISAreader.

FIG. 3 demonstrates that JES10-5A2 does not compete with the binding of228B/C-1-HRP to human IL13, indicating that 228B/C-1 and JES10-5A2 bindto different sites on human IL13.

Example 5 Screening for Anti-IL13 Neutralizing Monoclonal Antibodies byan IL-13-Autocrine Dependent Proliferation Assay Using L-1236 and HDLM-2Cells

L-1236 and HDLM-2 are Hodgkin lymphoma cell lines obtained from theGerman Collection of Microorganisms and Cell Cultures (DSMZ,Braunschweig, Germany). These cell lines produce IL13 which in turnactivates their cell proliferation in an autocrine fashion (Kapp U et.al., J. Exp. Med. 189:1939 (1999)).

Cells were cultured (25,000 cells/well) in the presence or absence ofdifferent anti-IL13 MAb (0.2, 0.02 and 0.002 μg/mL) in 5% CO₂ at 37° C.for 3-5 days. Cell proliferation was then measured either by an assayusing the tetrazolium compound MTS (Promega, Madison, Wis.) (readouts atOD₄₉₀) or by the incorporation of ³H-thymidine (Amersham Biosciences,Piscataway, N.J.).

The addition of an anti-IL13 neutralizing MAb to the culture of thesecell lines was expected to inhibit their proliferation by the bindingand inactivation of the IL13 produced by these cells. The resultsillustrated in FIG. 4 shows the effect of anti-IL13 MAb of the presentinvention on the proliferation of L-1235 cells. MAb 228B/C-1 displaysthe highest potency of inhibition of L-1236 cell proliferation in adose-dependent manner among the neutralizing antibodies tested. TA1-37(an anti-IL13 MAb generated by using E. coli expressed human IL13 asimmunogen) did not have any inhibitory activity even at a dose as highas 0.2 μg/mL. Similar results were obtained with HDLM-2 cells.

Example 6 Assay For IL13-Regulated CD14 And CD23 Expression on PrimaryHuman Monocytes

IL13 induces suppression of CD14 expression and the up-regulation ofCD23 expression in the human monocytes (de Waal Malefyt et al., J.Immunol., 151: 6370 (1993), Chomarat et al., Int. Rev. Immunol., 17: 1(1998)). Peripheral blood leukocytes (PBLs) were isolated from freshlycollected, heparinized whole blood of healthy human donors bydensity-gradient centrifugation in Histopaque-1077 (Sigma). PBLs(1.5×10⁶) suspended in RPMI-1640 medium (Invitrogen) with 5% fetalbovine serum were added to each well of a 96-well tissue culture platecontaining recombinant IL13 (final 10 ng/mL=0.813 nM) and an anti-IL13monoclonal antibody or an irrelevant antibody (three-fold serialdilutions, from a final 12 μg/mL=80 nM). CD14 expression or CD23expression on monocytes was suppressed or up-regulated, respectively, bythe addition of 0.813 nM human IL13 to the incubating medium. The mediumcontrol contained RPMI-1640/FBS medium without recombinant IL13.

The cells were incubated in 5% CO₂ at 37° C. for 2 days. The cells wereharvested for staining with anti-CD14-FITC or anti-CD23-PE (BDBiosciences-Pharmingen). The expression levels of CD14 and CD23 in themonocyte population were measured by flow cytometry and represented byMedian Fluorescence Intensity (MFI).

The effects of anti-IL13 MAbs on IL13-suppressed CD14 expression onhuman monocytes are depicted in FIG. 5. Among all the anti-IL13 MAbstested, 228B/C-1 had the highest potency in inhibiting the effect ofIL13 on CD14 expression. Complete inhibition of the effect of IL13 wasachieved at 0.33 nM. The inhibitory activities of MAbs 227-26 and 228A-4were moderate, whereas that of JES10-5A2 was weak. The effect of IL13could not be completely inhibited by JES10-5A2 even at 80 nM.

The effects of anti-IL13 MAbs on IL13-induced CD23 up-regulation onhuman monocytes are depicted in FIG. 6. Similar to the results on CD14expression (FIG. 5), 228B/C-1 was most potent in inhibiting the effectof IL13 on CD23 expression among the anti-IL13 MAbs tested. Completeinhibition by 228B/C-1 was achieved at 0.33 nM. The inhibitory potencyof JES10-5A2 was weak.

Based on the results presented in FIGS. 5 and 6, complete inhibition ofIL13 by 228B/C-1 can be achieved at a molar stoichiometric ratio of 1:2(MAb:IL13), and, therefore, 228B/C-1 is a very high affinityneutralizing MAb against human IL13.

Example 7 IL13-Induced STAT6 Phosphorylation Assay in THP-1 Cells

IL13 can activate the myeloid cell line THP-1 (ATCC, Manassas, Va.) toinduce phosphorylation of STAT6 which is a critical step in the signaltransduction pathway of IL13 (Murata T et al., Int. Immunol. 10:1103-1110 (1998). The anti-IL13 MAbs were tested for inhibition of IL13in this assay.

THP-1 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM)(Invitrogen) supplemented with 5% fetal bovine serum. On the day ofexperiments, the cells were washed and incubated in serum-free DMEM at37° C. in 5% CO₂ for 2 hours. 0.3×10⁶ cells in 80 μL of the serum-freemedium were then added to each well of a 96-well round-bottom plate. Onehundred and twenty microliters of medium containing human IL13 (finalconcentration of 10 ng/mL=0.813 nM) and anti-IL13 MAbs (5 fold serialdilutions, from final concentration of 0.5 μg/mL=3.333 nM). Negativecontrol wells containing either no IL13 or IL13 and an isotype-matchedirrelevant mouse MAb.

The mixtures were incubated at 37° C. under 5% CO₂ for 10 min. Theplates were then centrifuged at 300×g for 3 minutes at 4° C. Afterdiscarding the supernatant, the cell pellets were resuspended in 100 μLof Laemmli non-reducing sample buffer (SDS-PAGE loading buffer, BioRad,Calif.) and then transferred to microcentrifuge tubes. The tubes wereheated at 95° C. for 5 minutes and then centrifuged at 10,000×g for 10minutes at room temperature. The supernatants were collected andanalyzed by 4-20% gradient SDS-PAGE. The separated proteins weretransferred to PVDF membrane which was then incubated with diluted mouseanti-human Stat6 (Y641, phospho-specific) MAb (BD BiosciensesPharmingen).

The bound antibody was detected by HRP conjugated goat anti-mouse IgG(Fc) antibodies (Jackson ImmunoResearch Laboratories). Theimmunoreactive proteins were identified on film, using enhancedchemiluminescence detection (Supersignal West Pico ChemiluminescentSubstrate, Pierce)-FIG. 7 depicts the results of the effect of anti-IL13MAbs on IL13-induced phosphorylation of Stat6 in THP-1 cells. Stat6 isphosphorylated in THP-1 cells treated with 0.813 nM human IL13.Dose-dependent inhibition of Stat6 phosphorylation was found when thecells were treated with MAbs 228B/C-1, 228A-4, 227-26, 227-43 andJES10-5A2. MAb 228B/C-1 is the most potent neutralizing antibodies amongthe anti-IL13 MAbs tested. Complete inhibition by 228B/C-1 was achievedat a concentration between 0.667 nM and 0.133 nM. The approximate molarstoichiometric ratio between 228B/C-1 and IL13 for complete inhibitionwas 1:2. It is consistent with the data shown in FIGS. 5 and 6.

Example 8 Molecular Cloning of Heavy and Light Chain Genes EncodingAnti-IL13 Monoclonal Antibodies

Total RNA was isolated from hybridoma cells using a QIAGEN kit(Valencia, Calif.). Reverse transcription (first strand cDNA) reactionwas carried out as follows: 1-1.5 mg of total RNA was mixed with 1 ml 10mM dNTPs, 50 ng random Hexamers, and RNase free water in a final volumeof 12 mL.

The reaction mixture was incubated at 65° C. for 5 minutes and placed onice immediately for 1 minute. After a brief centrifugation, thefollowing reagents were added: 4 mL of 5× first strand buffer (250 mMTris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl₂), 2 mL of 0.1 mM DTT, and 1 mLof RNaseOUT RNase inhibitor (40 U/mL). After mixing, the reaction wasincubated at room temperature for 2 minutes. One milliliter ofSuperscript II RT (50 U/ml) was then added to the mixture for incubationat 25° C. for 10 minutes followed by 50 minutes at 42° C. After a briefcentrifugation, the reaction was incubated for 15 minutes at 70° C. toinactivate the reverse transcriptase. One microliter of RNase H (2 U/ml)was then added and the reaction was incubated for 20 minutes at 37° C.to destroy RNA.

To amplify the variable regions of the heavy and light chains, a methoddescribed by O'Brien and Jones (O'Brien S, and Jones T., “Humanizingantibodies by CDR grafting”, Antibody Engineering, Springer Lab manual,Eds. Kontermann and Duble, S (2001)) was used. Briefly, 5′ primers wereselected from the signal peptide region (11 sets for light chain and 12sets of degenerate primers for heavy chain) and 3′ primers were selectedfrom the constant region of either the light or heavy chain. 5′ and 3′primers (1.5 mL of 10 mM) were mixed with 5 mL of 10×PCR buffer (250 mMTris-HCl, pH 8.8, 20 mM MgSO₄, 100 mM KCl, 100 mM (NH₄)₂SO₄, 1% TritonX-100, 1 mg/mL nuclease free BSA), 1 mL cDNA as prepared previously, 1mL of Turbo pfu (Stratagene) and water to adjust the total volume of thereaction to 50 mL. PCR was performed as follows: 1 cycle at 94° C. for 4minutes; 25 cycles at 94° C. for 30 seconds, at 53° C. for 30 seconds,and at 72° C. for 45 seconds; and 1 cycle at 72° C. for 7 minutes.Reaction mixtures were resolved by electrophoresis in a 1% agarose gel.

Amplified DNA fragment was purified and cloned into a pcDNA3.1 vector.Cloning was carried out using the Invitrogen TOPO cloning kit followingthe manufacturers suggested protocol (Invitrogen). Fifteen to twentycolonies of transformed E. coli were used for plasmid purification.Plasmids were sequenced using a T7 primer. The predominant sequences forthe heavy and light chains were cloned into an M13 Fab expression vectorby hybridization mutagenesis (Glaser S. et al. Antibody Engineering(Oxford University Press, New York (1995)), Near R I, BioTechniques 12:88 (1992)). Binding properties of the expressed Fab were confirmed byELISA. FIGS. 8-10 depict the VH and VL chain amino acid sequences for228B/C, 228A-4, and 227-26, respectively.

Example 9 Humanization of Clone 228B/C

A. General Protocol

The variable regions of murine antibody 228B/C were cloned and sequencedas described in Example 8. A chimeric Fab in a phage vector wasconstructed as a control which combined the variable regions of themurine 228B/C and the constant region of the human kappa chain and theCH1 part of human IgG.

To begin the humanization process, a suitable v gene sequence selectedfrom known human germ line gene sequences was selected to provide theframework regions one to three (FM1-FM3), and a suitable J gene sequencewas selected to provide framework 4 (FM4) according to the criteriadescribed in WO04/070010 (incorporated herein by reference). Thistemplate may be chosen based on, e.g., its comparative overall length,the size of the CDRs, the amino acid residues located at the junctionbetween the framework and the CDRs, overall homology, etc. The templatechosen can be a mixture of more than one sequence or may be a consensustemplate.

Constructing an expression vector comprising the heavy and/or lightchain variants generated comprised the formulas:FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4  (i) andFRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4  (ii),wherein FRH1, FRH2, FRH3, FRH4, FRL1, FRL2, FRL3 and FRL4 represent thevariants of the framework template heavy chain and light chain sequenceschosen from the germ line templates and the CDRs represent those of theparent antibody. The differences between the murine parent antibody andthe selected human template sequences were determined to serve as abasis for generating a library of antibody Fabs. This library can begenerated for the light chain individually, and then the heavy chain orsimultaneously. Affinity maturation of the CDR regions may also beanalyzed simultaneously or sequentially with the humanization of theframework.

The library of variant Fabs was generated containing (1) the murineamino acid residue, (2) the amino acid residue from the chosen humangerm line gene, or optionally, (3) a randomly selected amino acid, ateach of the selected positions found to differ from the murine frameworksequence. The desired variants were generated by annealing overlappingoligonucleotides and then incorporating the chosen residue at theframework positions that were of interest. An amplification of theannealed product was done using two primers, one of which wasbiotin-labeled. The biotin tag was used for the purification of asingle-strand of the primer and this was used as a mutagenic oligo in aKunkel-based mutagenesis reaction using the vector of interest in aU-template format (Rosok, M. J., et al., (1996) Journal of BiologicalChemistry 271: 22611-22618). After annealing and elongating the plasmid,the reaction underwent digestion with a unique restriction enzyme, XbaI,which cleaves the original template but not the newly synthesizedmutated strand. The plasmid was electroporated into competent cells foramplification and mixed with a phage-competent E. coli cell-type forgeneration of phage particles. The plasmid constructs are able tosynthesize a Fab which is secreted into the supernatant. Individualplaques were selected and the antibody eluted for analysis.

The library was analyzed for quality and completeness. Upon sequencing arandom sampling of the library, the number of candidates selected thathad the correct insertion of the Vk (or Vh) region was determined. Thisnumber was used to determine the overall efficiency of the library. Oncethe library was established, the candidates were screened using afunctional ELISA-based assay to determine which candidates producedfunctional Fabs specific for IL-13. Those candidates demonstratingactivity for IL-13 comparable to the chimeric clone were assayed furtherfor reproducibility. Several of the candidates were sequenced todetermine how tolerant the targeted framework positions were forhumanization.

After the libraries were found to be representative, variants wereanalyzed for binding affinity, and those found to have comparable orgreater binding affinity than the chimeric control antibody weresequenced. If the isolates analyzed did not contain a residue from humangerm line gene at a chosen position in the framework, it was concludedthat the human amino acid residue was not tolerated at that position. Atthis point, if only the murine and human amino acids were tested,another Fab library could be made randomizing the amino acids at thepositions where human template residues were not found. Fabs withsuitable replacement residues (non murine) would then be selected andconverted into whole MAbs. In addition, consensus templates may be usedas the starting framework.

B. IL-13 Monoclonal Antibody Vk Humanization

Humanization of the variable region of the light chain (Vk) wasperformed first. However, one can begin with either chain or humanizeboth chains simultaneously. The human template chosen was Human Template2 and involved studying the effects on 9 residues close to the CDRswithin the light chain to determine if they could be humanized without aloss of functional activity. The positions that were studied on thelight chain for the second round of screening were 4, 9, 12, 73, 81, 82,83, 84, and 109.

A library was generated varying each of these positions with either themurine or the human template residue. Approximately 860 variants werescreened using a functional ELISA assay. Only 18 candidates demonstratedcomparable function to the chimeric clone. These candidates were assayedfurther. Six candidates of the 18 demonstrated a greater affinity forantigen compared to the chimeric clone, and these 6 were sequenced. Thesequencing results are presented in FIGS. 11A and B, and from theseresults, positions 4, 12 and 81 favor the murine residue.

C. Vh Humanization

In order to assess the contribution of the heavy chain frameworkresidues to the overall function of the candidate antibody, a librarywas established varying 10 positions within the human DP27 templateframework that differed from the murine parent, while maintaining themurine light chain. The library was generated using synthesizedoverlapping oligonucleotides for the Vh, and generation of the murine Vkusing PCR. The Vk and Vh were then inserted into the Fab expressionvector using mutagenesis and the library was then screened forfunctional Fabs. The complexity of the library was (2¹⁰/70%)×3=3840.

A total of 1056 candidates were screened, using a 96-well format ELISAassay. The candidates from this library that were chosen for sequencingwere those that yielded the highest values from the screen results. Fiveof these high activity candidates were sequenced to determine theirlevel of humanization and their sequences are presented in FIGS. 12A andB. From these results, three of the framework residues on the heavychain favored the murine residue (#24, 68 and 94).

The second framework studied was the human template NEW. A combinatoriallibrary was generated in which both the Vk and Vh were humanizedconcurrently. Nine residues on the Vk were varied between the murine andthe human residues and nine residues were also chosen for the Vh.Approximately 5200 candidates (55 96-well plates) were screened fromthis library. From the screen, approximately 300 candidates yieldedresults comparable to the chimeric clone. From this group, thirtycandidates were sequenced to determine the humanization level of thesefunctional clones.

The sequencing results for the light chains are presented in FIGS. 11Aand B. The heavy chain sequences are presented in FIGS. 12 A & B.Position 83 on the Vk had a high incidence for retaining the murineresidue, whereas several positions in the Vh template favored the murineresidue. In particular, position 94 retained the murine residue in 29out of 30 candidates screened. Although no candidates appear to havecompletely humanized frameworks, several variable regions which werehighly humanized in either the Vk or Vh will be used for furtherhumanization. The most humanized Vk was combined with the most humanizedVh to assay functional activity. (See FIG. 13.)

A second library which combined the framework residues of the Vk and Vhof interest was generated using DP27 as the heavy chain template and HT2as the light chain template. As described above, overlappingoligonucleotides were synthesized which contained the human frameworkwith either human or murine residues at each position in question. Theseoligos were mixed and then annealed to generate the complete variableregions. The regions were amplified through PCR and then made intosingle-stranded fragments. The fragments were phosphorylated and thenused in a mutagenesis reaction to incorporate the variable regions intothe M13-based vector. The library was then screened for functional Fabsthat were specific for IL-13 in an ELISA-based assay. The sequences forthe light chain and heavy chains are shown in FIGS. 11 C&D and 12C&D,respectively.

From the sequencing results, the Vk chain was able to tolerate humanresidues throughout, and thus this chain was fully humanized. For theheavy chain, two positions were intolerant of the human residues:position 24 and 94. Thus, the heavy chain variable region was ˜98%humanized.

D. Generation of Combinatorial Humanized Candidates

Since no candidate picked up from the screening of either of thelibraries was fully humanized, the humanization was engineered. A seriesof candidates were generated in which the desired humanization levelswere obtained. The most humanized Vk from the HT2 library was combinedwith the most humanized Vh from either the NEW or the DP27 libraries.These combinatorial candidates were then assayed to determine whichmaintained the specific function while carrying the highest humanizationlevel. The candidates chosen from HT2-NEW were HT2-NEW #73 for heavychain and HT2-NEW #115 for the light chain. The candidates chosen fromHT2-DP27 light chain were HT2-DP27 #89 and HT2-DP27 #144, and thecandidates for heavy chain were HT2-DP27 #123 and HT2-DP27 #276. ForHT2-DP27, constructs were made as follows: #89 Vk with #276 Vh and #89Vk with #123 Vh; #144 Vk with #276 Vh and #144 Vk with #123 Vh. Inaddition, one construct was made with #144 Vk DP27 with #73 Vh NEW todetermine whether NEW and DP27 interactions with the HT2 light chaindiffered.

These combinations were tested by ELISA to determine if there was anyfurther loss of function upon further humanization. For these assays,the antigen IL-13 was captured on the plate in a limiting amount. Theanti-IL13 Fabs were then added to the plate at a known concentration andtitrated down the plate at a 1:3 dilution. Binding was detected with asecondary antibody that is specific for Fab. FIG. 13 depicts thefunctional assay results. FIG. 13A—115Vk/73Vh; FIG. 13B—89Vk/276Vh; FIG.13C—144Vk/276Vh; and FIG. 13D—144Vk/73Vh. From these data, the observedresults suggested that the engineered combinations of humanized variableregions did not adversely affect the binding of the Fabs to the antigen.

Because the results in FIGS. 11 and 12 suggested that the HT2 lightchain could be fully humanized and all but 2 positions in DP27 (24 and94) could be humanized, the ideal humanized candidate was engineered inwhich the only 2 murine residues remained. Upon generation of thisparticular candidate, the clone was assayed in comparison with itsparent as well as the other candidates to determine if there was anyloss of function. From the data presented in FIG. 14A, this humanizedcandidate shows no significant loss of function with this high degree ofhumanization (89 Vk/276G). Humanization was also done for the HT2-NEWframework candidate. This candidate has a final humanization level of98%, as there are two murine residues that remain on the heavy chain.FIG. 14B depicts the ELISA results for this construct (115Vk/73Vh FL).

An attempt was made to further humanize 89Vk/276G by replacing the tworemaining murine residues. Upon mutating the positions to the humanresidues, the candidate clones were assayed by ELISA and compared to theparent. However, a significant loss of function was observed uponreplacing the murine residues with those of the chosen template.Therefore, another library was generated in which the two positions onthe Vh were randomized to allow for all possible amino acids at thesetwo positions. The candidates were screened using a functional ELISAassay and thirty candidates that yielded comparable results to theparent clone (89Vk/276G) were sequenced to determine which amino acidswere present at the targeted positions. A list of the candidates and theamino acids at the two positions is shown below.

Candi- 24 94 Candi- 24 94 228B/C V G RL49 A T date date DP27 F R RL59 IM RL19 S L RL84 L T 89/ V G RL61 S T RL27 G V RL88 L S 276G RL7 A S RL62T T RL32 G G RL89 L S RL8 L S RL70 S L RL35 S L RL91 G L RL11 T V RL72 VT RL36 G V RL95 I L RL12 I I RL78 I M RL40 L S RL97 T T RL15 L L RL79 VT RL45 T T RL18 S R Candi- 24 94 Candi- 24 94 date date

Thus, from this screen, there are several amino acids which apparentlyare tolerated at the designated positions and yet do not result insignificant loss of function. Thus, by changing the framework residuesto amino acids that are not found in the murine sequence nor in thehuman framework, a fully functional Fab was generated withoutdetrimental effect on binding to the target antigen. The candidates thatwere further tested from this random library were RL-19 and RL-36.

Example 10 CDR Optimization

Upon determining the optimal framework sequence for the candidateanti-IL-13 antibody, optimization of the CDRs was performed. For thisprocess, the CDR amino acid sequence was randomized and then thelibraries were screened to identify those candidates which had equal orbetter functional activity than the parent clone. For this library, theparent candidate was RL-36 (see above). The six CDRs were randomized,one position at a time and the libraries were screened using afunctional ELISA. Strongly reactive candidates were sequenced forcomparison with the parent CDR. It is noted that all unique sequenceslisted in the tables below also appear in FIG. 20 with appropriate SEQID NO identifiers.

A CDR-L1 Optimization

CDR-L1 comprised 15 amino acids. Each of these positions was randomizedusing synthesized oligonucleotides which were the mixed in equimolaramounts to be used in a mutagenesis reaction. The efficiency ofincorporation of the mutagenic oligonucleotides was determined to be40%. Using this percentage, the number of candidates which needed to bescreened was 3600. The clones were assayed using a functional ELISA andthose clones that yielded comparable functional activity were sequenced.From the number of candidates that were screened, 166 positivecandidates were identified. From this group, 10 candidates weresequenced to determine the changes within the CDR. From the sequencingresults shown below results, the positions 11 and 14 lead to improvedaffinity are N to Q and M to L.

SEQ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ID NO: CDR-L1 R A S K S V D S  YG N S F M H 99 L1-21 R A S K S V D S Y G N S F L H 103 L1-39 R A S K S VD S Y G Q S F M H 100 L1-47 K A S K S V D S Y G N S F M H 194 L1-50 R AS K S V D S Y G N S Y M H 102 L1-59 R A S K S V D S Y G Q S F M H 100L1-61 R A S K S V D S Y G N S F M H 99 L1-62 R A S K S V D S Y G N S F LH 103 L1-63 R A S K S V D S Y G N S F L H 103 L1-117 N A S K S V D S Y GN S F M H 195 L1-125 R A S K S V D S Y G N S F M H 99

B. CDR2-L2 Optimization

CDR-L2 comprised 7 amino acids. This library was prepared as describedabove. The efficiency of this library was 80% and 840 clones wereassayed. The number of positive clones identified from the assay was 75and 11 were sequenced. From the results shown below, several positionswithin this CDR yielded improved activity, although the positions andreplacement amino acids appeared random. This result supports theobservation that CDR-L2 is farthest from the antigen binding site and assuch should exert the least influence upon antigen binding.

SEQ 1 2 3 4 5 6 7 ID NO: CDR-L2 L A S N L E S 104 L2-10 L A S N L N S105 L2-13 L A S N L E S 104 L2-25 L A S N L Q S 106 L2-37 L A T N L E S107 L2-41 L A S N L K S 108 L2-44 L A S N L E K 109 L2-45 L A S R L E S110 L2-53 L A S N L H S 111 L2-58 L A S N L S S 112 L2-65 L A S F L E S113 L2-70 L A N N L E S 114

C. CDR-L3 Optimization

CDR-L3 was composed of 9 amino acids. This library upon generationyielded an efficiency of 50%, requiring ˜1700 clones be screened. Fromthis screen, 257 positive candidates were identified and ten weresequenced. From these results, only one position yielded a change fromthe parent sequence. Several candidates demonstrated the same sequencewhich suggested that this positional change was highly favored (N to A).

SEQ 1 2 3 4 5 6 7 8 9 ID NO: CDR-L3 Q Q N N E D P R T 115 L3-1 Q Q N N ED P R T 115 L3-32 Q Q N A E D P R T 116 L3-90 Q Q N N E D P R T 115L3-100 Q Q N N E D P R T 115 L3-150 Q Q N N E D P R T 115 L3-170 Q Q N AE D P R T 116 L3-185 Q Q N A E D P R T 116 L3-207 Q Q N A E D P R T 116L3-225 Q Q N N E D P R T 115

D. CDR-H1 Optimization

CDR-H1 comprised 5 amino acids. The efficiency of this library was 80%,requiring only about 600 candidates be screened. From the screen, therewere 138 positive clones and eleven of the clones were sequenced. Fromthe results are listed below, the second position within this CDR seemedto offer the greatest chance of improvement of antigen binding. However,several amino acids favorably affect binding.

SEQ 1 2 3 4 5 ID NO: CDR-H1 A Y S V N 117 H1-2 A K S V N 118 H1-12 G Y SV N 120 H1-18 A K S V N 118 H1-24 A K S V N 118 H1-31 A H S V N 121H1-89 A Y S V N 117 H1-90 G Y S V N 120 H1-114 A S S V N 185 H1-115 A HS V N 121 H1-123 A R S V N 122 H1-126 A R S V N 122

E. CDR-H2 Optimization

CDR-H2 comprised 16 amino acids. The efficiency of this library was 70%,which meant that over 2100 candidates needed to be screened. From thescreen, 192 positive candidates were identified and thirteen weresequenced to determine the changes that occurred within the CDR. Fromthe sequencing results listed below, several positions improved bindingaffinity but none of the amino acid changes appeared significantlydifferent from the parent.

SEQ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 ID NO: CDR-H2 M I W G D G K IV Y N S A L K S 123 H2-38 M I W G D G K I S Y N S A L K S 124 H2-43 M IW G D G K I V Y N S A L E S 125 H2-51 M I W G D G K I V Y N S A L K S126 H2-66 M I W G D G K I S Y N S A L K S 124 H2-79 M I W G D G K I V YN S D L K S 127 H2-86 M I W G D G K V V Y N S A L K S 128 H2-101 M I W GD G K I V Y N S E L K S 129 H2-109 M I W G D G K I A Y N S A L K S 130H2-119 M I W G D G K I V Y N S A L K E 131 H2-121 M V W G D G K I V Y NS A L K S 132 H2-129 M I W G D G K I V Y N S A L K S 123 H2-169 M I W GD G K I V Y N S A L A S 133 H2-176 M I W G D G K K V Y N S A L K S 134

F. CDR-H3 Optimization

CDR-H3 comprised 10 amino acids. This CDR in general is believed to bethe one that imposes the greatest influence on antigen binding, becausethis loop is generally in the middle of the binding site. This libraryhad an efficiency of 40%, and so 2400 candidates needed to be screened.Of these, 174 positive candidates were identified and ten were sequencedto determine the changes within the CDR. The results listed belowindicated that the change from Y to R in the third position may be animportant one for improvement in binding.

SEQ 1 2 3 4 5 6 7 8 9 10 ID NO: H3 D G Y Y P Y A M D N 135 H3-1 D G R YP Y A M D N 136 H3-30 D G Y Y P Y A M S N 139 H3-73 D G Y Y P Y A M A N140 H3-89 D G Y Y P Y A M A N 140 H3-130 D G R Y P Y A M D N 136 H3-131D G R Y P Y A M D N 136 H3-133 D G Y Y P Y A L D N 141 H3-135 D G R Y PY A M D N 136 H3-161 D G Y Y P Y A M D N 135 H3-162 D G Y Y P Y A M K N137

G. Combinatorial Library

Once the changes within the CDRs which yielded the greatest overallimprovement in antigen binding were determined, the best candidates werethen combined to see if these changes improved binding. Thus, acandidate was engineered to combine all favorable amino acidsubstitutions.

To generate the combinatorial library, the initial clone was the onethat incorporated the alteration in CDR-L1-59 (N to Q). To this clone,the other changes were made for CDR-L3, N to A (position 4), for CDR-H1,Y to either R, H, K or S (position 2), for CDR-H3, Y to R (position 3)and D to either K or S (position 9). No changes were made to CDR-L2 orCDR-H2. Over 1100 candidates from this library were screened using afunctional ELISA assay. A total of 120 candidates were identified ashaving activity greater than the parent clone. The sequences of thoseclones are shown in FIG. 15.

To confirm that these combinatorial candidates maintained function, acompetition assay was performed. For this assay, IL-13 was captured onan ELISA plate. The candidates, which are purified Fabs, were pre-mixedin varying concentrations to a constant concentration of labeledchimeric anti-IL-13 Fab. This mixture was added to the ELISA plate. Thelabeled chimeric anti-IL-13 capable of binding to the plate-bound IL-13were detected.

From the results of this competition, the two candidates assayeddemonstrated equivalent ability to compete with the chimeric candidate(228 B/C #3) for binding to IL-13 (FIG. 16). The irrelevant Fab is 5I,which demonstrates no ability to compete. FIG. 17 depicts the sequencesof three affinity matured candidates.

Example 11 Epitope Mapping

Anti-IL13 MAb 228B/C-1 binds to a conformational epitope and binds tocynomologous monkey IL13 with the same high affinity as it does to humanIL13. However, 228B/C does not bind to murine IL13. So, the strategydevised for epitope mapping was to exchange small portions of the monkeyIL13 with the corresponding mouse IL13 sequence. Overlappingoligonucleotides were synthesized as shown in FIG. 18. Two rounds of PCRwere performed to assemble the IL13 hybrid constructs so that part ofmonkey IL13 was replaced by the corresponding sequence from mouse IL13(FIG. 18). The final PCR amplified IL13 coding regions were cloned intopcDNA3.1 vector in frame with a V5 tag using TOPO cloning kit(Invitrogen). All PCR amplified region were confirmed by sequencing tocontain only the desired domain swapping mutations and not additionalunwanted mutation in the expression vectors.

The anti-IL13 MAb binding epitope was identified as a 8-mer peptide fromamino acid #49 to 56, ESLINVSG (SEQ ID NO 18). This epitope is locatedin Helix-B and loop-BC in human IL13. When the epitope peptide derivedfrom cyno-IL13 was used to swap the corresponding sequence in murineIL13, the resulting hybrid IL13 molecule can bind to 228B/C withaffinity similar to that of the original cynoIL13, further validatedthat 228B/C MAb binding to cyno or human IL13 at this peptide betweenresidual #49-56. Sequence comparison between human, cyno, and murineIL13 reveals only three residues Ile52, Val54, Gly56 in human IL13 arenot conserved, suggesting the critical residues for IL13 and anti-IL13MAb interaction through this 8-mer peptide is determined by one orcombination of some of these three residues.

This epitope was further confirmed by peptide spot analysis. The entirehuman IL13 peptide was scanned with a series of overlapping 12-merpeptides synthesized via SPOT on cellulose membrane. The only anti-IL13MAb reactive peptide was identified as a 12-mer peptide of amino acid#44-56, YCAALESLINVS (SEQ ID NO 19), which is overlapping with theregion identified through domain swapping experiments.

Deposits

The following cultures have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas Va. 20110-2209USA (ATCC):

Hybridoma ATCC NO. Deposit Date Anti-IL13 228B/C-1 PTA-5657 Nov. 20,2003 Anti-IL13 228A-4 PTA-5656 Nov. 20, 2003 Anti-IL13 227-26 PTA-5654Nov. 20, 2003 Anti-IL13 227-43 PTA-5655 Nov. 20, 2003

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture for 30 years fromthe date of deposit. The organism will be made available by ATCC underthe terms of the Budapest Treaty, which assures permanent andunrestricted availability of the progeny of the culture to the publicupon issuance of the pertinent U.S. patent.

The assignee of the present application has agreed that if the cultureon deposit should die or be lost or destroyed when cultivated undersuitable conditions, it will be promptly replaced on notification with aviable specimen of the same culture. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cultures deposited, sincethe deposited embodiments are intended as illustration of one aspect ofthe invention and any culture that are functionally equivalent arewithin the scope of this invention. The deposit of material herein doesnot constitute an admission that the written description hereincontained is inadequate to enable the practice of any aspect of theinvention, including the best mode thereof, nor is it to be construed aslimiting the scope of the claims to the specific illustration that itrepresents. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and fall within thescope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The invention claimed is:
 1. A method for treating asthma in a human patient, comprising administering to a human patient diagnosed with asthma an effective amount of a monoclonal anti-interleukin-13 (“IL-13”) antibody that specifically binds human IL-13, wherein the anti-IL-13 antibody comprises a heavy chain variable region and a light chain variable region comprising the complementarity determining regions of an antibody produced by the hybridoma designated with American Type Culture Collection (“ATCC”) accession number PTA-5657, thereby treating asthma in the patient.
 2. A method for treating asthma in a human patient, comprising administering to a human patient diagnosed with asthma an effective amount of a monoclonal anti-IL-13 antibody that specifically binds human IL-13, wherein the anti-IL-13 antibody comprises a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2 and CDRH3 having the amino acid sequences of SEQ ID NO: 117, SEQ ID NO: 123, and SEQ ID NO: 135, respectively; and wherein the anti-IL-13 antibody comprises a light chain variable region comprising complementarity determining regions CDRL1, CDRL2 and CDRL3 having the amino acid sequences of SEQ ID NO: 99, SEQ ID NO: 104, and SEQ ID NO: 115, respectively, thereby treating asthma in the patient.
 3. The method of claim 1, wherein the heavy chain variable region comprises complementarity determining regions CDRH1, CDRH2 and CDRH3 having the amino acid sequences of SEQ ID NO: 117, SEQ ID NO: 123, and SEQ ID NO: 135, respectively.
 4. The method of claim 1, wherein the light chain variable region comprises complementarity determining regions CDRL1, CDRL2 and CDRL3 having the amino acid sequences of SEQ ID NO: 99, SEQ ID NO: 104, and SEQ ID NO: 115, respectively.
 5. The method of claim 1, wherein the anti-IL-13 antibody further comprises human framework regions.
 6. The method of claim 2, wherein the anti-IL-13 antibody comprises the amino acid sequence of SEQ ID NO: 142, and the amino acid sequence of SEQ ID NO:
 143. 7. The method of claim 2, wherein the anti-IL-13 antibody is an IgG antibody.
 8. The method of claim 2, wherein the anti-IL-13 antibody is an IgG1, an IgG2, an IgG3 or an IgG4 antibody.
 9. The method of claim 1, wherein the anti-IL-13 antibody is humanized.
 10. The method of claim 2, wherein the anti-IL-13 antibody is humanized.
 11. The method of claim 1, wherein the anti-IL-13 antibody is a monovalent antibody, a multispecific antibody, a chimeric antibody, a single chain antibody, a Fab fragment, or a F(ab′) fragment.
 12. The method of claim 2, wherein the anti-IL-13 antibody is a monovalent antibody, a multispecific antibody, a chimeric antibody, a single chain antibody, a Fab fragment, or a F(ab′) fragment.
 13. The method of claim 1, wherein the anti-IL-13 antibody is a bispecific antibody.
 14. The method of claim 2, wherein the anti-IL-13 antibody is a bispecific antibody.
 15. The method of claim 6, wherein the anti-IL-13 antibody is a bispecific antibody.
 16. The method of claim 10, wherein the anti-IL-13 antibody is a bispecific antibody.
 17. The method of claim 1, wherein the anti-IL-13 antibody is conjugated to a molecule.
 18. The method of claim 2, wherein the anti-IL-13 antibody is conjugated to a molecule.
 19. The method of claim 2, 6, or 10, wherein the effective amount is between 0.1 mg/kg and 20 mg/kg.
 20. The method of any one of claims 1, 2, 9, 10, 13, and 14, wherein the asthma is allergic asthma.
 21. The method of any one of claims 1, 2, 9, 10, 13, and 14, wherein the asthma is non-allergic (intrinsic) asthma. 